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Abstract: Biodefense vaccines are developed against a diverse group of pathogens. Vaccines were developed for some of these pathogens a long time ago but they are facing new challenges to move beyond the old manufacturing technologies. New vaccines to be developed against other pathogens have to determine whether to follow traditional vaccination strategies or to seek new approaches. Advances in basic immunology and recombinant DNA technology have fundamentally transformed the process of formulating a vaccine concept, optimizing protective antigens, and selecting the most effective vaccine delivery approach for candidate biodefense vaccines. [Copyright &y& Elsevier]

The V3 loop in the HIV envelope gp120 is one of the immunogenic sites targeted by Abs. The V3 crown in particular has conserved structural elements recognized by cross-reactive neutralizing Abs, indicating its potential contribution in protection against HIV. Crystallographic analyses of anti-V3 crown mAbs in complex with the V3 peptides have revealed that these mAbs recognize the conserved sites on the V3 crown via two distinct strategies: a cradle-binding mode (V3C) and a ladle-binding (V3L) mode. However, almost all of the anti-V3 crown mAbs studied in the past were isolated from chronically HIV-infected individuals. The extents to which the two types of anti-V3 crown Abs are generated by vaccination are unknown. This study analyzed the prevalence of V3C-type and V3L-type Ab responses in HIV-infected individuals and in HIV envelope-immunized humans and animals using peptide mimotopes that distinguish the two Ab types. The results show that both V3L-type and V3C-type Abs were generated by the vast majority of chronically HIV-infected humans, although the V3L-type were more prevalent. In contrast, only one of the two V3 Ab types was elicited in vaccinated humans or animal models, irrespective of HIV-1 envelope clades, envelope constructs (oligomeric or monomeric), and protocols (DNA plus protein or protein alone) used for vaccinations. The V3C-type Abs were produced by vaccinated humans, macaques, and rabbits, whereas the V3L-type Abs were made by mice. The V3C-type and V3L-type Abs generated by the vaccinations were able to mediate virus neutralization. These data indicate the restricted repertoires and the species-specific differences in the functional V3-specific Ab responses induced by the HIV envelope vaccines. The study implies the need for improving immunogen designs and vaccination strategies to broaden the diversity of Abs in order to target the different conserved epitopes in the V3 loop and, by extension, in the entire HIV envelope. [ABSTRACT FROM AUTHOR]

Recombinant protein vaccines are commonly formulated with an immune-stimulatory compound, or adjuvant, to boost immune responses to a particular antigen. Recent studies have shown that, through recognition of molecular motifs, receptors of the innate immune system are involved in the functions of adjuvants to generate and direct adaptive immune responses. However, it is not clear to which degree those receptors are also important when the adjuvant is used as part of a novel heterologous prime-boost immunization process in which the priming and boosting components are not the same type of vaccines. In the current study, we compared the immune responses elicited by a pentavalent HIV-1 DNA prime–protein boost vaccine in mice deficient in either Toll-like receptor 4 (TLR4) or myeloid differentiation primary response gene 88 (MyD88) to wildtype mice. HIV gp120 protein administered in the boost phase was formulated with either monophosphoryl lipid A (MPLA), QS-21, or Al(OH) 3 . Endpoint antibody titer, serum cytokine response and T-cell memory response were assessed. Neither TLR4 nor MyD88 deficiency had a significant effect on the immune response of mice given vaccine formulated with QS-21 or Al(OH) 3 . However, TLR4- and MyD88-deficiency decreased both the antibody and T-cell responses in mice administered HIV gp120 formulated with MPLA. These results further our understanding of the activation of TLR4 and MyD88 by MPLA in the context of a DNA prime/protein boost immunization strategy. [ABSTRACT FROM AUTHOR]

Highlights: [•] We constructed the rAdV-SjTPI.opt adenoviral vector based vaccine and tested its efficacy against Schistosoma japonicum infection. [•] Intramuscular rAdV-SjTPI.opt induced Th1 biased immune responses in the host, while subcutaneous rAdV-SjTPI.opt induced Th2 predominant immune responses. [•] Oral rAdV-SjTPI.opt induced low levels of immune responses and no significant protection. [•] Intramuscular rAdV-SjTPI.opt provided a consistent and repeatable higher protective effect in mice. [•] Associated higher levels of specific Th1, antibody responses and lower level of IL-17A may contribute to the higher protective effect. [Copyright &y& Elsevier]

Abstract: Yersinia pestis (Y. pestis) is the causative pathogen of plague, a highly fatal disease for which an effective vaccine, especially against mucosal transmission, is still not available. Like many bacterial infections, antigen-specific antibody responses have been traditionally considered critical, if not solely responsible, for vaccine-induced protection against Y. pestis. Studies in recent years have suggested the importance of T cell immune responses against Y. pestis infection but information is still limited about the details of Y. pestis antigen-specific T cell immune responses. In current report, studies are conducted to identify the presence of CD8+ T cell epitopes in LcrV protein, the leading antigen of plague vaccine development. Furthermore, depletion of CD8+ T cells in LcrV DNA vaccinated Balb/C mice led to reduced protection against lethal intranasal challenge of Y. pestis. These findings establish that an LcrV DNA vaccine is able to elicit CD8+ T cell immune responses against specific epitopes of this key plague antigen and that a CD8+ T cell immune response is involved in LcrV DNA vaccine-elicited protection. Future studies in plague vaccine development will need to examine if the presence of detectable T cell immune responses, in particular CD8+ T-cell immune responses, will enhance the protection against Y. pestis in higher animal species or humans. [Copyright &y& Elsevier]

Abstract: Gene-based hepatitis B virus (HBV) vaccines have been proposed as a novel approach to improve the immunogenicity toward non-responders and to allow for protection against potential viral escape mutants. Furthermore, there is significant interest in using DNA or viral vector vaccines to serve as therapeutic agents to treat chronic HBV infections that are resistant to existing drug therapies. However, the key protective antigen of HBV, the surface protein (HBsAg), can be expressed in three different sizes due to its multiple translational initiation sites: small, middle, and large forms of HBsAg. It is not clear whether the immunogenicity of these HBsAg is same, especially their ability to elicit HBsAg-specific B cell and T cell immune responses in addition to the traditional serum HBsAg-specific antibody responses. In the current study, the immunogenicity of three forms of HBsAg DNA vaccines was analyzed individually in a mouse model. Our results indicated that different forms of the HBsAg have unique immunogenicity profiles and this information is useful for the selection of optimal gene-based HBV vaccines for further improved prophylactic and therapeutic applications. [ABSTRACT FROM AUTHOR]

Abstract: Schistosomiasis is an endemic, zoonotic parasitic disease caused by Schistosoma japonicum that remains a public health concern and an effective vaccine is needed. Triose-phosphate isomerase from S. japonicum is a promising schistosome vaccine antigen shown to be immunogenic when delivered as a DNA vaccine, however, the previous S. japonicum triose-phosphate isomerase (SjTPI) DNA vaccine needs to be further optimized to achieve higher protection. In the current study, codon optimization of SjTPI DNA insert, combined with electroporation but not with the addition of a tPA leader or heat-shock protein in-frame with the SjTPI gene insert, enhanced Th1-type antibody and cytokine production and most significantly, achieved great than 50% reduction of infection against challenge with S. japonicum cercariae, a major milestone in S. japonicum vaccine development. Our results suggest that the combination of a codon optimized vaccine design and an efficient vaccine delivery system can greatly improve the potential of a SjTPI DNA vaccine as a viable schistosome vaccine candidate. [Copyright &y& Elsevier]

Abstract: Hand, foot, and mouth disease (HFMD) is a common viral illness in infants and children caused by viruses that belong to the enterovirus genus of the picornavirus family. Although most HFMD do not result in serious complications, outbreaks of HFMD caused by enterovirus 71 (EV71) can present with a high rate of neurological complications, including meningoencephalitis, pulmonary complications, and possibly death. HFMD caused by EV71 has become a major emerging infectious disease in Asia and the highly pathogenic potential of EV71 clearly requires the attention of world medical community. Although vaccine development for EV71 is active and ongoing in Asian countries, a greater joint effort is needed for vaccine researchers and developers in both developed and developing countries to produce a safe and effective EV71 vaccine. [Copyright &y& Elsevier]

Abstract: Using a gp120 envelope glycoprotein from the JR-FL strain of human immunodeficiency virus-1 (HIV-1) as a model antigen, the goal of the current study was to evaluate the level and quality of antibody responses elicited by different prime-boost vaccination regimens (protein only, DNA only, DNA plus protein) in rabbits. Our data demonstrated that incorporating DNA immunization as a prime in a heterologous prime-boost regimen was able to elicit a more diverse and conformational epitope profile, higher antibody avidity, and improved neutralizing activity than immunization with only protein. Additionally, this improved neutralizing activity was observed in spite of similar antibody specificities and avidities seen when only DNA vaccination was used, providing additional evidence that the use of a combination immunization regimen increases the protective antibody response. Insights gained from the current study confirmed that the heterologous DNA prime-protein boost approach is effective in eliciting not only high level but also improved quality of antigen-specific antibody responses, and thus may offer a new technology platform to develop better and safer subunit vaccines. [Copyright &y& Elsevier]

Abstract: The use of a DNA immunization approach to deliver protective antigens against Yersinia pestis (Y. pestis) has been successful in previously reported studies. In the current study, the gene designs for V and F1, two well-studied virulent factors serving as main targets for vaccine development, were altered to explore additional options in hopes of improving the protective immunity of DNA vaccines expressing these two antigens. Compared to the wild type V gene DNA vaccines, the use of codon optimized V gene sequences was effective in improving the antigen expression, titers of anti-V antibody responses, and survival against a mucosal lethal challenge. For the F1 DNA vaccine, removal of the N-terminal hydrophobic region was able to improve protective immunity. However, adding a mammalian signal peptide sequence to F1 actually led to reduced protection despite it inducing slightly higher anti-F1 antibody responses. The F1 gene can be fused with a gene coding for YscF, a newly confirmed partial protective antigen for Y. pestis, to produce DNA vaccines that express fused F1 and YscF antigens. One design, in particular, that had YscF fused to the downstream sequence of F1, produced better protection than separate F1 or YscF DNA vaccines, suggesting a potential synergistic effect between these two antigens. Findings from the above studies indicated that there are multiple approaches to optimize the protective immunity for plague DNA vaccines. Most importantly, proper antigen engineering to produce optimal antigen gene inserts in DNA vaccines can clearly play a major role in the future designs of a wide range of DNA vaccines. [Copyright &y& Elsevier]

Abstract: Developing an effective HIV-1 vaccine will require strategies to enhance antigen presentation to the immune system. In a previous study we demonstrated a marked increase in immunogenicity of the highly glycosylated HIV-1 gp120 protein following enzymatic addition of α-gal epitopes to the carbohydrate chains. In the present study we determined whether gp120αgal can also serve as an effective platform for targeting other HIV-1 proteins to APC and thus increase immunogenicity of both proteins. For this purpose we produced a recombinant fusion protein between gp120 and the HIV-1 matrix p24 protein (gp120/p24). Multiple α-gal epitopes were synthesized enzymatically on the gp120 portion of the fusion protein to generate a gp120αgal/p24 vaccine. Immune responses to gp120αgal/p24 compared to gp120/p24 vaccine lacking α-gal epitopes were evaluated in α1,3galactosyltransferase knockout (KO) mice. These mice lack α-gal epitopes and, therefore, are capable of producing the anti-Gal antibody. T cell responses to p24, as assessed by ELISPOT and by CD8+ T cells intracellular staining assays for IFNγ, was on average 12- and 10-fold higher, respectively, in gp120αgal/p24 immunized mice than in mice immunized with gp120/p24. In addition, cellular and humoral immune responses against gp120 were higher by 10–30-fold in mice immunized with gp120αgal/p24 than in gp120/p24 immunized mice. Our data suggest that the α-gal epitopes on the gp120 portion of the fusion protein can significantly augment the immunogenicity of gp120, as well as that of the fused viral protein which lacks α-gal epitopes. This strategy of anti-Gal mediated targeting to APC may be used for production of effective HIV-1 vaccines comprised of various viral proteins fused to gp120. [Copyright &y& Elsevier]

Abstract: Neutralizing antibody is associated with the prevention and clearance of influenza virus infection. Microneutralization (MN) and hemagglutination inhibition (HI) assays are currently used to evaluate neutralizing antibody responses against human and avian influenza viruses, including H5N1. The MN assay is somewhat labor intensive, while HI is a surrogate for neutralization. Moreover, use of replication competent viruses in these assays requires biosafety level 3 (BSL-3) containment. Therefore, a neutralization assay that does not require BSL-3 facilities would be advantageous. Toward this goal, we generated a panel of pseudotypes expressing influenza hemagglutinin (HA) and neuraminidase (NA) and developed a pseudotype-based neutralization (PN) assay. Here we demonstrate that HA/NA pseudotypes mimic release and entry of influenza virus and that the PN assay exhibits good specificity and reveals quantitative difference in neutralizing antibody titers against different H5N1 clades and subclades. Using immune ferret sera, we demonstrated excellent correlation between the PN, MN, and HI assays. Thus, we conclude that the PN assay is a sensitive and quantifiable method to measure neutralizing antibodies against diverse clades and subclades of H5N1 influenza virus. [Copyright &y& Elsevier]

Abstract: Cholera is a potentially lethal diarrhea disease caused by the gram-negative bacterium Vibrio cholerae. The need for an effective cholera vaccine is clearly indicated but the challenges of eliciting both systemic and mucosal immune responses remains a significant challenge. In the current report, we discovered that a DNA vaccine expressing a protective cholera antigen, cholera toxin B subunit (CTB), delivered parenterally can elicit both systemic and mucosal anti-CTB antibody responses in mice. The priming effect by DNA immunization was demonstrated by higher mucosal antibody responses following one boost with the inactivated cholera vaccine (KWC-B) delivered orally when compared to the twice oral administration of KWC-B alone. This finding indicates that DNA vaccines delivered parenterally are effective in eliciting mucosal protective immune responses—a unique advantage for DNA vaccination that has not yet been well realized and should bring value to the development of novel vaccination approaches against mucosally transmitted diseases. [Copyright &y& Elsevier]

Abstract: Hepatitis B Virus (HBV) infection remains a major worldwide infectious disease with serious long-term morbidity and mortality. The limited selections of drug treatment are not able to control the progress of disease in people with active and persistent HBV infection. Immunotherapy to control the degree of viral infection is one possible alternative solution to this challenge. HBV DNA vaccines, with their strong ability to induce cell-mediated immune responses, offer an attractive option. HBV surface protein is important in viral immunity. Re-establishing anti-S immunity in chronic HBV infected patients will bring significant benefit to the patients. Previous studies have shown that HBV S DNA vaccines are immunogenic in a number of animal studies. In the current study, we further investigated the effect of glycosylation to the expression and immunogenicity of S DNA vaccines. Our results demonstrate that deglycosylation at the two potential N-linked glycosylation sites in S protein resulted in a significant decrease of S-specific cell-mediated immune responses, but did not affect anti-S antibody responses. This finding provides important direction to the development of S DNA vaccines to elicit the optimal and balanced antibody and cell-mediated immune responses to treat people with HBV chronic infections. [Copyright &y& Elsevier]

Abstract: This report describes the safety observations following administration of a polyvalent DNA prime–protein boost HIV-1 vaccine formulated with adjuvant QS21. Local injection site reactions were the most common (65% of subjects), and included type IV delayed-type hypersensitivity (DTH) reactions at prior DNA inoculation sites in 12 of 28 (43%) subjects following protein vaccination. Systemic reactions revealed two cases of vasculitis temporally related to inoculation with recombinant Env protein+QS21 adjuvant. Questions remain regarding the cause of the vasculitis, but the unique DTH observation may have contributed to the high level of immune responses previously reported for this vaccine. [Copyright &y& Elsevier]

Abstract: An optimally effective AIDS vaccine would likely require the induction of both neutralizing antibody and cell-mediated immune responses, which has proven difficult to obtain in previous clinical trials. Here we report on the induction of human immunodeficiency virus type-1 (HIV-1)-specific immune responses in healthy adult volunteers that received the multi-gene, polyvalent, DNA prime–protein boost HIV-1 vaccine formulation, DP6-001, in a Phase I clinical trial. Robust cross-subtype HIV-1-specific T cell responses were detected in IFN-γ ELISPOT assays. Furthermore, we detected high titer serum antibody responses that recognized a wide range of primary HIV-1 Env antigens and also neutralized pseudotyped viruses that express the primary Env antigens from multiple HIV-1 subtypes. These findings demonstrate that the DNA prime–protein boost approach is an effective immunization method to elicit both humoral and cell-mediated immune responses in humans, and that a polyvalent Env formulation could generate broad immune responses against HIV-1 viruses with diverse genetic backgrounds. [Copyright &y& Elsevier]

Abstract: The trivalent inactivated vaccine (TIV) is used to prevent seasonal influenza virus infection in humans, however, the immunogenicity of this vaccine may be influenced by the priming effect of previous influenza vaccinations or exposure to antigenically related influenza viruses. The current study examines the immunogenicity of a clinically licensed TIV in rabbits naïve to influenza antigens. Animals were immunized with either the licensed TIV, a bivalent (H1 and H3) HA DNA vaccine or the combination of both. Temporal and peak level serum anti-influenza virus IgG responses were determined by enzyme-linked immunosorbent assay (ELISA). Functional antibody responses were measured by hemagglutination inhibition and microneutralization against either A/NewCaledonia//20/99 (H1N1) or A/Panama/2007/99 (H3N2) influenza viruses. Our results demonstrate that the immunogenicity of the TIV is low in sero-negative animals. More significantly, the heterologous DNA prime-TIV boost regimen was more immunogenic than the homologous prime-boost using either TIV or DNA vaccines alone. This finding justifies further investigation of HA DNA vaccines as a priming immunogen for the next generation of vaccines against seasonal or pandemic influenza virus infections. [Copyright &y& Elsevier]

Summary: Immunogenicity of DNA vaccines varies significantly due to many factors including the inherent immunogenicity of the protein antigen encoded in the DNA vaccine, the optimal immune responses that can be achieved in different animal models and in humans with different genetic backgrounds and, to a great degree, the delivery methods used to administer the DNA vaccines. Based on published results, only the gene gun-mediated delivery approach has been able to elicit protective levels of immune responses in healthy, adult volunteers by DNA immunization alone without the use of another vaccine modality as a boost. Recent results from animal studies suggest that electroporation is also effective in eliciting high level immune responses. However, there have been no reports to identify the similarities and differences between these two leading physical delivery methods for DNA vaccines against infectious disease targets. In the current study, we compared the relative immunogenicity of a DNA vaccine expressing a hemagglutinin (HA) antigen from an H5N1 influenza virus in two animal models (rabbit and mouse) when delivered by either intramuscular needle immunization (IM), gene gun (GG) or electroporation (EP). HA-specific antibody, T cell and B cell responses were analyzed. Our results indicate that, overall, both the GG and EP methods are more immunogenic than the IM method. However, EP and IM stimulated a Th-1 type antibody response and the antibody response to GG was Th-2 dominated. These findings provide important information for the further selection and optimization of DNA vaccine delivery methods for human applications. [Copyright &y& Elsevier]

Summary: Yersinia Pestis outer proteins, plasminogen activator protease and Yop secretion protein F are necessary for the full virulence of Yesinia pestis and have been proposed as potential protective antigens for vaccines against plague. In the current study, we used DNA immunization as a tool to study the relative protective immunity of these proteins with a standardized intranasal challenge system in mice. While the natural full-length gene sequences for most of these Y. pestis proteins did not display a good level of protein expression in vitro when delivered by a DNA vaccine vector, the overall immunogenicity of these wild type gene DNA vaccines was low in eliciting antigen-specific antibody responses and gene sequence modifications improved both of these parameters. However, even modified YopD, YopO and YscF antigens were only able to partially protect immunized mice at various levels against lethal challenge with Y. pestis KIM 1001 strain while no protection was observed with either the YopB or Pla antigens. These results demonstrate that DNA immunization is effective in screening, optimizing and comparing optimal antigen designs and immunogenicity of candidate antigens for the development of a subunit-based plague vaccine. [Copyright &y& Elsevier]

Summary: An optimally effective AIDS vaccine would likely require the induction of both neutralizing antibody and cell-mediated immune responses, which has proven difficult to obtain in previous clinical trials. Here we report on the induction of Human Immunodeficiency Virus Type-1 (HIV-1)-specific immune responses in healthy adult volunteers that received the multi-gene, polyvalent, DNA prime–protein boost HIV-1 vaccine formulation, DP6-001, in a Phase I clinical trial conducted in healthy adult volunteers of both genders. Robust cross-subtype HIV-1-specific T cell responses were detected in IFNγ ELISPOT assays. Furthermore, we detected high titer serum antibody responses that recognized a wide range of primary HIV-1 Env antigens and also neutralized pseudotyped viruses that express the primary Env antigens from multiple HIV-1 subtypes. These findings demonstrate that the DNA prime–protein boost approach is an effective immunization method to elicit both humoral and cell-mediated immune responses in humans, and that a polyvalent Env formulation could generate broad immune responses against HIV-1 viruses with diverse genetic backgrounds. [Copyright &y& Elsevier]

Abstract: Human cytomegalovirus (HCMV) glycoprotein complex II (gcII) consists of two glycoproteins, gM and gN. Although gcII specific IgG purified from HCMV positive patient sera can neutralize HCMV, there has been no report describing the generation of virus-neutralizing antibodies by immunization with individual recombinant gM or gN antigens. In the current study, gM and gN antigens were expressed by the mammalian expression vector pJW4303 and used as DNA vaccines to determine the immunogenicity of these proteins. Sera from mice or rabbits immunized with individual or combinations of gM and gN DNA vaccines contained gM and gN specific antibodies as confirmed by ELISA and Western blot analyses. The combined gM and gN antigens induced the strongest antibody responses that recognized both gM and gcII complex while gM DNA vaccine alone could only elicit antibody specific for gM antigen. When given alone, the gN DNA vaccine did not induce detectable gcII specific antibody even though in vitro gN expression was confirmed by the formation of gM/gN complex in FSK cells using a gN-specific monoclonal antibody 14-16A. The neutralizing antibody titer of anti-gM/gN sera (1:128) was higher than that of anti-gM sera (1:32) against the autologous virus, HCMV AD169. Heterologous HCMV strains including Towne and Davis could also be neutralized by the anti-gM/gN antisera. Our data supported the rationale for the use of the HCMV gM/gN protein complex as protective antigens for subunit based HCMV vaccine development. DNA vaccination is an effective approach to express the gM/gN antigen complex in vivo without the need to express and purify these highly insoluble and structurally complicated antigens. [Copyright &y& Elsevier]

Abstract: Because of the high failure rate of antibiotic treatment in patients with anthrax there is a need for additional therapies such as passive immunization with therapeutic antibodies. In this study, we used codon-optimized plasmid DNAs (DNA vaccines) encoding Bacillus anthracis protective antigen (PA) to immunize rabbits for producing anti-anthrax antibodies for use in passive immunotherapy. The antisera generated with these DNA vaccines were of high titer as measured by ELISA. The antisera were also able to protect J774 macrophage cells by neutralizing the cytotoxic effect of exogenously added anthrax lethal toxin, and of the toxin released by B. anthracis (Sterne strain) spores following infection. In addition, the antisera passively protected mice against pulmonary challenge with an approximate 50 LD50 dose of B. anthracis (Sterne strain) spores. The protection in mice was obtained when the antiserum was given 1h before or 1h after challenge. We further demonstrated that IgG and F(ab′)2 components purified from anti-PA rabbit hyperimmune sera retained similar levels of neutralizing activities against both exogenously added B. anthracis lethal toxin and toxin produced by B. anthracis (Sterne strain) spores. The high titer antisera we produced will enable an immunization strategy to supplement antibiotic therapy for improving the survival of patients with anthrax. [Copyright &y& Elsevier]

Abstract: Optimized antigen expression is critical to the immunogenicity of DNA vaccines. A number of approaches have been proposed to enhance the antigen expression and/or immunogenicity of DNA vaccines, but their relative contributions have not been compared in a same antigen system. In the current study, optimization of codon usage, enhancement of viral promoter function and selection of secretary leader sequences were evaluated for their roles in improving the immunogenicity of a same model antigen, the HIV-1 envelope glycoprotein. Our data demonstrated that all these factors can work synergistically to improve the final antigen expression and immunogenicity of HIV-1 Env DNA vaccines, indicating they work through different mechanisms. The best result came from the approach that optimized all three components in a DNA vaccine design. Our study further revealed that the levels of HIV-1 env-specific RNA transcripts in transiently transfected 293T cells were higher from the codon-optimized gene than the wild type counterpart. This finding suggested other mechanism may also contribute to the increased antigen expression and immunogenicity of codon-optimized DNA vaccines in addition to the improved tRNA usage in mammalian cells for codon-optimized viral genes as previously reported. [Copyright &y& Elsevier]

Abstract: A toxicity and immunogenicity study, evaluating the safety of a polyvalent DNA prime/protein boost HIV-1 vaccine (DP6-001), was examined in rabbits. Animals were primed with a cocktail of six different DNA plasmids expressing five HIV-1 env genes and one gag gene followed by boosting with five gp120 proteins homologous to the DNA vaccines. The vaccine was shown to be immunogenic as evident from the induction of high-titered anti-Env and anti-Gag antibodies. There was an absence of detectable adverse effects on key toxicology parameters. Although plasmids persisted in the injection sites following single administration for 64 days, no evidence of integration into the host genomic DNA was observed. These studies demonstrate that a novel polyvalent DNA prime/protein boost vaccine lacks signs of toxicity and DNA integration in a rabbit model, and immunogenicity and toxicology data support clinical testing of the vaccine in humans. [Copyright &y& Elsevier]

There is an urgent need to develop effective vaccines against pneumonic plague, a highly lethal and contagious disease caused by the Gram-negative bacterium Yersinia pestis. Here we demonstrate that a novel DNA vaccine expressing a modified V antigen (LcrV) of Y. pestis, with a human tissue plasminogen activator (tPA) signal sequence, elicited strong V-specific antibody responses in BALB/c mice. This tPA-V DNA vaccine protected mice from intranasal challenge with lethal doses of Y. pestis. In comparison, a DNA vaccine expressing the wild type V antigen was much less effective. Only tPA-V formed oligomers spontaneously, and elicited a higher IgG2a anti-V antibody response in immunized mice, suggesting increased TH1 type cellular immune response. Our data indicate that antigen engineering is effective in inducing high quality protective immune responses against conformationally sensitive antigens. These results support that optimized DNA vaccines have the potential to protect against bacterial pathogens than is generally recognized. [Copyright &y& Elsevier]

The ability to elicit humoral and cell-mediated immune (CMI) responses from DNA immunization by combinational use of codon optimization and C3d component of complement was evaluated in this study. DNA vaccines that express either the wild type or the codon optimized gp120 gene coding for the envelope (Env) glycoprotein of human immunodeficiency virus (HIV-1) from the primary isolate JR-FL strain were compared to the same forms fused to three tandem copies of the murine C3d genes. Either codon optimization or C3d fusion alone was effective at generating early appearance, higher binding and neutralizing antibody responses. We also observed that cell-mediated immune responses against HIV Env could also be enhanced by C3d fusion. However, for both humoral and CMI responses, there were no synergistic effects when the combination of codon optimization and C3d fusion was employed. [Copyright &y& Elsevier]