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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. [ABSTRACT FROM AUTHOR]

Nearly a third of the human population is at risk of infection with the four serotypes of dengue viruses, and it is estimated that more than 100 million infections occur each year. A licensed vaccine for dengue viruses has become a global health priority. A major challenge to developing a dengue vaccine is the necessity to produce fairly uniform protective immune responses to all four dengue virus serotypes. We have developed two bivalent dengue virus vaccines, using a complex adenovirus vector, by incorporating the genes expressing premembrane (prM) and envelope (E) proteins of dengue virus types 1 and 2 (dengue-1 and -2, respectively) (CAdVax-Den12) or dengue-3 and -4 (CAdVax-Den34). Rhesus macaques were vaccinated by intramuscular inoculation of a tetravalent dengue vaccine formulated by combining the two bivalent vaccine constructs. Vaccinated animals produced high-titer antibodies that neutralized all four serotypes of dengue viruses in vitro. The ability of the vaccine to induce rapid, as well as sustained, protective immune responses was examined with two separate live-virus challenges administered at 4 and 24 weeks after the final vaccination. For both of these virus challenge studies, significant protection from viremia was demonstrated for all four dengue virus serotypes in vaccinated animals. Viremia from dengue-1 and dengue-3 challenges was completely blocked, whereas viremia from dengue-2 and dengue-4 was significantly reduced, as well as delayed, compared to that of control-vaccinated animals. These results demonstrate that the tetravalent dengue vaccine formulation provides significant protection in rhesus macaques against challenge with all four dengue virus serotypes. [ABSTRACT FROM AUTHOR]

Abstract: DNA shuffling and screening technologies were used to produce chimeric DNA constructs expressing antigens that shared epitopes from all four dengue serotypes. Three shuffled constructs (sA, sB and sC) were evaluated in the rhesus macaque model. Constructs sA and sC expressed pre-membrane and envelope genes, whereas construct sB expressed only the ectodomain of envelope protein. Five of six, and four of six animals vaccinated with sA and sC, respectively, developed antibodies that neutralized all 4 dengue serotypes in vitro. Four of six animals vaccinated with construct sB developed neutralizing antibodies against 3 serotypes (den-1, -2 and -3). When challenged with live dengue-1 or dengue-2 virus, partial protection against dengue-1 was observed. These results demonstrate the utility of DNA shuffling as an attractive tool to create tetravalent chimeric dengue DNA vaccine constructs, as well as a need to find ways to improve the immune responses elicited by DNA vaccines in general. [Copyright &y& Elsevier]

Abstract: We employed DNA shuffling and screening technologies to develop a single recombinant dengue envelope (E) antigen capable of inducing neutralizing antibodies against all four antigenically distinct dengue serotypes. By DNA shuffling of codon-optimized dengue 1–4 E genes, we created a panel of novel chimeric clones expressing C-terminal truncated E antigens that combined epitopes from all four dengue serotypes. DNA vaccines encoding these novel chimeras induced multivalent T cell and neutralizing antibody responses against all four dengue serotypes in mice. By contrast, a mixture of four unshuffled, parental DNA vaccines failed to produce tetravalent neutralizing antibodies in mice. The neutralizing antibody titers for some of these antigens could be further improved by extending the sequences to express full-length pre-membrane and envelope proteins. The chimeric antigens also protected mice against a lethal dengue-2 virus challenge. These data demonstrate that DNA shuffling and associated screening can lead to the selection of multi-epitope antigens against closely related dengue virus serotypes and suggest a broad utility for these technologies in optimizing vaccine antigens. [Copyright &y& Elsevier]

A dengue-1 DNA vaccine containing sequences encoding premembrane and envelope proteins (DIME) was previously shown to elicit virus neutralizing antibodies in rhesus and Aotus monkeys, and the primates were partially protected from viremia upon challenge. To increase the neutralizing antibody levels and subsequent protection from virus challenge, four strategies were evaluated: (a) coimmunization with a plasmid expressing Aotus GM-CSF gene; (b) coimmunization with a plasmid containing human immunostimulatory sequences (ISS); (c) coimmunization with both the GM-CSF gene and ISS; and (d) delivery of vaccine using the needle-free Biojector system. Vaccination with the mixed formulation containing DIME, GM-CSF gene, and ISS, by either needle injection or Biojector, led to neutralizing antibody titers that were stable for up to 6 months after vaccination. Furthermore, 6 of 7 monkeys (85%), and 7 of 8 monkeys (87%) receiving this formulation were completely protected from viremia when challenged 1 and 6 months after vaccination, respectively. This is a significant improvement compared to our previous study in which one of three monkeys (33%) receiving just the DIME vaccine was completely protected from viremia at 6 months after immunization. [Copyright &y& Elsevier]

A dengue 2 plasmid DNA vaccine (pD2) expressing the pre-membrane and envelope proteins (preM-E) was modified by replacing the dengue transmembrane and cytoplasmic sequences with those of the mouse lysosome-associated membrane protein (pD2/LAMP). Immunofluorescence and confocal microscopy of human 293, NIH 3T3, and macrophage IC21 cell lines transfected with pD2/LAMP showed that the preM-E/LAMP protein chimera was present in vesicles containing endogenous LAMP and major histocompatability complex class II (MHC II), in contrast to the non-vesicular localization of native preM-E protein lacking the LAMP targeting sequence. Mice immunized with pD2 showed an antigen-specific immunoglobulin response but the neutralizing antibodies titers (plaque reduction neutralization test, PRNT50) elicited by the native protein were minimal. In contrast, vaccination with pD2/LAMP resulted in PRNT50 of 270, 320 and 160 at approximately 1, 3 and 8 months after two immunizations with 50 μg DNA, and approached 100% neutralization at 1:20 dilution. Additional immunization with pD2/LAMP, after 8 months, increased the neutralizing antibody titers to >640. Comparable neutralizing antibody responses were induced by two vector backbones, pVR1012 and pVax-1, at 5 and 50 μg of DNA. The neutralizing responses to the pD2/LAMP chimera were greatly superior to those elicited by pD2 in all conditions. These results underscore the importance of MHC class II presentation of DNA-encoded dengue-virus envelope protein for production of neutralizing antibodies. [Copyright &y& Elsevier]

Abstract: A dengue (DEN) virus type 3 DNA vaccine expressing pre-membrane and envelope genes was tested for immunogenicity and protective efficacy in Aotus monkeys. Five of six vaccinated animals demonstrated moderate DEN-specific antibody responses as measured by ELISA and virus neutralization in vitro. By contrast, none of the six control animals developed detectable anti-DEN antibodies. When five vaccinated animals were challenged with live DEN-3 virus and viremia determined by PCR amplification of viral RNA in serum samples, one animal was completely protected and two were partially protected as indicated by a decrease in mean days of viremia. The results demonstrate the ability of the DEN-3 DNA vaccine to elicit a neutralizing antibody response and to partially protect against live virus challenge. These findings support the inclusion of this construct in a tetravalent DNA vaccine. [Copyright &y& Elsevier]

• Two-step purification of Dengue virus using Chromatographic methods. • Psoralen-inactivation of Dengue virus. • Evaluation of psoralen-inactivated Dengue virus vaccines in mice. • Evaluation of psoralen-inactivated Dengue virus vaccines in nonhuman primates. Dengue fever, caused by dengue viruses (DENV 1–4) is a leading cause of illness and death in the tropics and subtropics. Therefore, an effective vaccine is urgently needed. Currently, the only available licensed dengue vaccine is a chimeric live attenuated vaccine that shows varying efficacy depending on serotype, age and baseline DENV serostatus. Accordingly, a dengue vaccine that is effective in seronegative adults, children of all ages and in immunocompromised individuals is still needed. We are currently researching the use of psoralen to develop an inactivated tetravalent dengue vaccine. Unlike traditional formalin inactivation, psoralen inactivates pathogens at the nucleic acid level, potentially preserving envelope protein epitopes important for protective anti-dengue immune responses. We prepared highly purified monovalent vaccine lots of formalin- and psoralen-inactivated DENV 1–4, using Capto DeVirS and Capto Core 700 resin based column chromatography. Tetravalent psoralen-inactivated vaccines (PsIV) and formalin-inactivated vaccines (FIV) were prepared by combining the four monovalent vaccines. Mice were immunized with either a low or high dose of PsIV or FIV to evaluate the immunogenicity of monovalent as well as tetravalent formulations of each inactivation method. In general, the monovalent and tetravalent PsIVs elicited equivalent or higher titers of neutralizing antibodies to DENV than the FIV dengue vaccines and this response was dose dependent. The immunogenicity of tetravalent dengue PsIVs and FIVs were also evaluated in nonhuman primates (NHPs). Consistent with what was observed in mice, significantly higher neutralizing antibody titers for each dengue serotype were observed in the NHPs vaccinated with the tetravalent dengue PsIV compared to those vaccinated with the tetravalent dengue FIV, indicative of the importance of envelope protein epitope preservation during psoralen inactivation of DENV. [ABSTRACT FROM AUTHOR]

Background The WHO estimates that there may be 50 million cases of dengue virus (DENV) infection worldwide every year. There is no safe vaccine against DENV licensed in the United States. The development of a balanced and effective anti-DENV vaccine is vital to preventing morbidity and mortality. Codagenix used its proprietary SAVE (Synthetic Attenuated Virus Engineering) platform to generate and test a live attenuated, tetravalent vaccine against DENV. Methods Codagenix used SAVE to substitute under-represented human codons and codon-pairs into the E protein sequences of contemporary strains of DENV1-4, producing either a fully human-cell-deoptimized prM-E (E-Min), or a partially deoptimized prM-E (E-W/Min) to allow for balancing of the vaccine's immunogenicity. Full genomes containing deoptimized E-Min and E-W/Min in the DENV2 backbone were transfected into cells to recover live-attenuated, human-cell-deoptimized vaccine strains. Mice were vaccinated with 106 FFU of each DENV vaccine (alone or together), boosted on day 21 and assessed for neutralizing antibodies by PRNT50 and survival after lethal challenge with mouse-adapted wild-type (WT) DENV. Cynomolgus macaques were immunized with a mixture of 106 FFU of each DENV vaccine strain. Two doses were administered on study day 1 and 57 and serum neutralizing antibodies were determined on day 57 and 85 by a microneutralization assay. Results SAVE deoptimized DENV viruses grew to wild-type (between 107 and 108 FFU/ml) levels at permissive temperatures (<37C). All vaccine strains generated neutralizing antibody levels comparable to WT. A tetravalent formulation containing all four E-Min strains protected mice from lethal challenge with DENV3. A tetravalent formulation of Codagenix DENV-E-W/Min vaccine elicited a robust and balanced neutralizing antibody response in non-human primates (NHPs) against all four DENV serotypes after a single dose. A second vaccine dose did not boost antibody titers significantly. Conclusion The ability to rationally balance the attenuation of multiple vaccine strains, thereby avoiding antibody-dependent enhancement, is a unique advantage of the Codagenix SAVE platform. Codagenix DENV vaccine viruses generated balanced, sterilizing immunity in NHPs after one dose. Disclosures All authors: No reported disclosures. [ABSTRACT FROM AUTHOR]

Phase 1 clinical trials with a DNA vaccine for dengue demonstrated that the vaccine is safe and well tolerated, however it produced less than optimal humoral immune responses. To determine if the immunogenicity of the tetravalent dengue DNA vaccine could be enhanced, we explored alternate, yet to be tested, methods of vaccine administration in non-human primates. Animals were vaccinated on days 0, 28 and 91 with either a low (1 mg) or high (5 mg) dose of vaccine by the intradermal or intramuscular route, using either needle-free injection or electroporation devices. Neutralizing antibody, IFN-γ T cell and memory B cell responses were compared to a high dose group vaccinated with a needle-free intramuscular injection delivery device similar to what had been used in previous preclinical and clinical studies. All previously untested vaccination methodologies elicited improved immune responses compared to the high dose needle-free intramuscular injection delivery group. The highest neutralizing antibody responses were observed in the group that was vaccinated with the high dose formulation via intradermal electroporation. The highest IFN-γ T cell responses were also observed in the high dose intradermal electroporation group and the CD8+ T cells were the dominant contributors for the IFNγ response. Memory B cells were detected for all four serotypes. More than a year after vaccination, groups were challenged with dengue-1 virus. Both the low and high dose intradermal electroporation groups had significantly fewer days of dengue-1 virus RNAemia compared to the control group. The results from this study demonstrate that using either an electroporation device and/or the intradermal route of delivery increases the immune response generated by this vaccine in non-human primates and should be explored in humans. [ABSTRACT FROM AUTHOR]

Background: Middle East respiratory syndrome (MERS) is a severe respiratory illness with an overall mortality of 35%. There is no licensed or proven treatment. Passive immunotherapy approaches are being developed to prevent and treat several human medical conditions where alternative therapeutic options are absent. We report the safety of a fully human polyclonal IgG antibody (SAB-301) produced from the hyperimmune plasma of transchromosomic cattle immunised with a MERS coronavirus vaccine.Methods: We did a phase 1 double-blind, placebo-controlled, single-dose escalation trial at the National Institutes of Health Clinical Center. We recruited healthy participants aged 18-60 years who had normal laboratory parameters at enrolment, a body-mass index of 19-32 kg/m2, and a creatinine clearance of 70 mL/min or more, and who did not have any chronic medical problems that required daily oral medications, a positive rheumatoid factor (≥15 IU/mL), IgA deficiency (<7 mg/dL), or history of allergy to intravenous immunoglobulin or human blood products. Participants were randomly assigned by a computer-generated table, made by a masked pharmacist, to one of six cohorts (containing between three and ten participants each). Cohorts 1 and 2 had three participants, randomly assigned 2:1 to receive active drug SAB-301 versus normal saline placebo; cohorts 3 and 4 had six participants randomised 2:1; and cohorts 5 and 6 had ten participants, randomised 4:1. Participants received 1 mg/kg, 2·5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 50 mg/kg of SAB-301, or equivalent volume placebo (saline control), on day 0, and were followed up by clinical, laboratory, and pharmacokinetic assessments on days 1, 3, 7, 21, 42, and 90. The primary outcome was safety, and immunogenicity was a secondary outcome. We analysed the intention-to-treat population. This trial is registered with ClinicalTrials.gov, number NCT02788188.Findings: Between June 2, 2016, and Jan 4, 2017, we screened 43 participants, of whom 38 were eligible and randomly assigned to receive SAB-301 (n=28) or placebo (n=10). 97 adverse events were reported: 64 adverse events occurred in 23 (82%) of 28 participants receiving SAB-301 (mean 2·3 adverse events per participant). 33 adverse events occurred in all ten participants receiving placebo (mean 3·3 adverse events per participant). The most common adverse events were headache (n=6 [21%] in participants who received SAB-301 and n=2 [20%] in those receiving placebo), albuminuria (n=5 [18%] vs n=2 [20%]), myalgia (n=3 [11%] vs n=1 [10%]), increased creatine kinase (n=3 [11%] vs 1 [10%]), and common cold (n=3 [11%] vs n=2 [20%]). There was one serious adverse event (hospital admission for suicide attempt) in one participant who received 50 mg/kg of SAB-301. The area under the concentration-time curve (AUC) in the 50 mg/kg dose (27 498 μg × days per mL) is comparable to the AUC that was associated with efficacy in a preclinical model.Interpretation: Single infusions of SAB-301 up to 50 mg/kg appear to be safe and well tolerated in healthy participants. Human immunoglobulin derived from transchromosomic cattle could offer a new platform technology to produce fully human polyclonal IgG antibodies for other medical conditions.Funding: National Institute of Allergy and Infectious Diseases, National Institutes of Health, and Biomedical Advanced Research and Development Authority. [ABSTRACT FROM AUTHOR]

This study describes an antibody-dependent NK cell degranulation assay, as a biomarker to assess antibody-dependent cellular cytotoxicity (ADCC) response in influenza plasma and for antibody therapies against influenza infection. The concentration of neutralizing antibodies (NAbs) against the hemagglutinin receptor of influenza viruses is a current determinant in protection against infection, particularly following receipt of the seasonal influenza vaccine. However, this is a limited assessment of protection, because: (i) NAb titers that incur full protection vary; and (ii) NAb titers do not account for the entire breadth of antibody responses against viral infection. Previous reports have indicated that antibodies that prime ADCC play a vital role in controlling influenza infections, and thus should be quantified for assessing protection against influenza. This report demonstrates a non-radioactive assay that assesses NK cell activation as a marker of ADCC, in which NK cells interact with opsonized viral antigen expressed on the surface of infected Raji target cells resulting in effector cell degranulation (surrogate CD107a expression). A positive correlation was determined between HAI titers and sustained NK cell activation, although NK cell activation was seen in plasma samples with HAI titers below 40 and varied amongst samples with high HAI titers. Furthermore, sustained NK cell degranulation was determined for influenza-vaccinated transchromosomic bovine intravenous immunoglobulin, indicating the potential utility of this therapy for influenza treatment. We conclude that this assay is reproducible and relevant. [ABSTRACT FROM AUTHOR]

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne RNA virus that causes low mortality but high morbidity rates in humans. In addition to natural outbreaks, there is the potential for exposure to VEEV via aerosolized virus particles. There are currently no FDA-licensed vaccines or antiviral therapies for VEEV. Passive immunotherapy is an approved method used to protect individuals against several pathogens and toxins. Human polyclonal antibodies (PAbs) are ideal, but this is dependent upon serum from convalescent human donors, which is in limited supply. Non-human-derived PAbs can have serious immunoreactivity complications, and when "humanized," these antibodies may exhibit reduced neutralization efficiency. To address these issues, transchromosomic (Tc) bovines have been created, which can produce potent neutralizing human antibodies in response to hyperimmunization. In these studies, we have immunized these bovines with different VEEV immunogens and evaluated the protective efficacy of purified preparations of the resultant human polyclonal antisera against low- and high-dose VEEV challenges. These studies demonstrate that prophylactic or therapeutic administration of the polyclonal antibody preparations (TcPAbs) can protect mice against lethal subcutaneous or aerosol challenge with VEEV. Furthermore, significant protection against unrelated coinfecting viral pathogens can be conferred by combining individual virus-specific TcPAb preparations. [ABSTRACT FROM AUTHOR]

We explored the feasibility of collecting convalescent plasma for passive immunotherapy of Middle East respiratory syndrome coronavirus (MERS-CoV) infection by using ELISA to screen serum samples from 443 potential plasma donors: 196 patients with suspected or laboratory-confirmed MERS-CoV infection, 230 healthcare workers, and 17 household contacts exposed to MERS-CoV. ELISA-reactive samples were further tested by indirect fluorescent antibody and microneutralization assays. Of the 443 tested samples, 12 (2.7%) had a reactive ELISA result, and 9 of the 12 had reactive indirect fluorescent antibody and microneutralization assay titers. Undertaking clinical trials of convalescent plasma for passive immunotherapy of MERS-CoV infection may be feasible, but such trials would be challenging because of the small pool of potential donors with sufficiently high antibody titers. Alternative strategies to identify convalescent plasma donors with adequate antibody titers should be explored, including the sampling of serum from patients with more severe disease and sampling at earlier points during illness. [ABSTRACT FROM AUTHOR]

Background In outbreak settings, mass vaccination strategies could maximize health protection of military personnel. Self-administration of live attenuated influenza vaccine (LAIV) may be a means to vaccinate large numbers of people and achieve deployment readiness while sparing the use of human resources. Methods A phase IV, open-label, randomized controlled trial evaluating the immunogenicity and acceptance of self-administered (SA) LAIV was conducted from 2012 to 2014. SA subjects were randomized to either individual self-administration or self-administration in a group setting. Control randomized subjects received healthcare worker-administered (HCWA) LAIV. Anti-hemagglutinin (HAI) antibody concentrations were measured pre- and post-vaccination. The primary endpoint was immunogenicity non-inferiority between SA and HCWA groups. Subjects were surveyed on preferred administration method. Results A total of 1077 subjects consented and were randomized (529 SA, 548 HCWA). Subject characteristics were very similar between groups, though SA subjects were younger, more likely to be white and on active duty. The per-protocol analysis included 1024 subjects (501 SA, 523 HCWA). Post-vaccination geometric mean titers by vaccine strain and by study group (HCWA vs. SA) were: A/H1N1 (45.8 vs. 48.7, respectively; p = 0.43), A/H3N2 (45.5 vs. 46.4; p = 0.80), B/Yamagata (17.2 vs. 17.8; p = 0.55). Seroresponses to A components were high (∼67%), while seroresponses to B components were lower (∼25%). Seroresponse did not differ by administration method. Baseline preference for administration method was similar between groups, with the majority in each group expressing no preference. At follow-up, the majority (64%) of SA subjects preferred SA vaccine. Conclusions LAIV immunogenicity was similar for HCWA and SA vaccines. SA was well-tolerated and preferred to HCWA among those who performed SA. [ABSTRACT FROM AUTHOR]

Four distinct serotypes of dengue viruses (DENV) are the cause of re-emerging dengue fever (DF) and dengue hemorrhagic fever (DHF). Dengue circulation in the Caribbean has gone from none or single serotype to multiple serotypes co-circulating with reports of continuing cycles of progressively more severe disease in the region. Few studies have investigated dengue on Sint Eustatius. Blood samples were collected to determine the prevalence of antibodies against dengue in the Sint Eustatius population. Greater than 90% of the serum samples (184 of 204) were positive for anti-flavivirus antibodies by enzyme linked immunosorbance assay (ELISA). Plaque reduction neutralization test (PRNT), specific for dengue viruses, showed that 171 of these 184 flavivirus antibody positive sera had a neutralization titer against one or more DENV serotypes. A majority of the sera (62%) had neutralizing antibody to all four dengue serotypes. Only 26 PRNT positive sera (15%) had monotypic dengue virus neutralizing antibody, most of which (20 of 26) were against DENV2. Evidence of infection with all four serotypes was observed across all age groups except in the youngest age group (10–19 years) which contained only DENV2 positive individuals. In a multiple logistic regression model, only the length of residence on the island was a predictor of a positive dengue PRNT50 result. To our knowledge this is the first dengue serosurveillance study conducted on Sint Eustatius since the 1970s. The lack of antibodies to the DEN1, 3, and 4 in the samples collected from participants under 20 years of age suggests that only DEN2 has circulated on island since the early 1990s. The high prevalence of antibodies against dengue (83.8%) and the observation that the length of time on the island was the strongest predictor of infection suggests dengue is endemic on Sint Eustatius and a public health concern that warrants further investigation. [ABSTRACT FROM AUTHOR]

Abstract: A prototype dengue-1 DNA vaccine was shown to be safe and immunogenic in a previous Phase 1 clinical trial. Anti-dengue-1 neutralizing antibody responses were detectable only in the group of volunteers receiving the high dose of nonadjuvanted vaccine and the antibody titers were low. Vaxfectin®, a lipid-based adjuvant, enhances the immunogenicity of DNA vaccines. We conducted a nonhuman primate study to evaluate the effect of Vaxfectin® on the immunogenicity of a tetravalent dengue DNA vaccine. Animals were immunized on days 0, 28 and 84, with each immunization consisting of 3mg of Vaxfectin®-adjuvanted tetravalent dengue DNA vaccine. The use of Vaxfectin® resulted in a significant increase in anti-dengue neutralizing antibody responses against dengue-1, -3 and -4. There was little to no effect on T cell responses as measured by interferon gamma ELISPOT assay. Animals immunized with the Vaxfectin®-formulated tetravalent DNA vaccine showed significant protection against live dengue-2 virus challenge compared to control animals (0.75 mean days of viremia vs 3.3 days). Animals vaccinated with nonadjuvanted DNA had a mean 2.0 days of viremia. These results support further evaluation of the Vaxfectin®-adjuvanted tetravalent dengue DNA vaccine in a Phase 1 clinical trial. [Copyright &y& Elsevier]

Abstract: Candidate dengue DNA vaccine constructs for each dengue serotype were developed by incorporating pre-membrane and envelope genes into a plasmid vector. A Phase 1 clinical trial was performed using the dengue virus serotype-1 (DENV-1) vaccine construct (D1ME100). The study was an open-label, dose-escalation, safety and immunogenicity trial involving 22 healthy flavivirus-naïve adults assigned to one of two groups. Each group received three intramuscular injections (0, 1, and 5 months) of either a high dose (5.0mg, n =12) or a low dose (1.0mg, n =10) DNA vaccine using the needle-free Biojector® 2000. The most commonly reported solicited signs and symptoms were local mild pain or tenderness (10/22, 45%), local mild swelling (6/22, 27%), muscle pain (6/22, 27%) and fatigue (6/22, 27%). Five subjects (41.6%) in the high dose group and none in the low dose group developed detectable anti-dengue neutralizing antibodies. T-cell IFN gamma responses were detected in 50% (4/8) and 83.3% (10/12) of subjects in the low and high dose groups, respectively. The safety profile of the DENV-1 DNA vaccine is acceptable at both doses administered in the study. These results demonstrate a favorable reactogenicity and safety profile of the first in human evaluation of a DENV-1 DNA vaccine. [ABSTRACT FROM AUTHOR]

Background: The hemagglutination-inhibition (HAI) assay is a critical component for measurement of immunogenicity in influenza vaccine development. It is unknown if the results can be influenced by sample type and anticoagulants. The purpose of this study was to evaluate the influence of different sample collection methods, in particular different anticoagulants, and choice of plasma or serum, on influenza virus serological assays.Methods: Blood samples from thirty donors previously immunized against influenza viruses were collected using six different types of blood collection tubes, two of which collect serum and four of which contain various anticoagulants for collecting plasma. Serum: (1) serum separator tubes (SST); and (2) Plus Plastic serum "red-top serum" tubes. Plasma: (3) spray-coated K2 ethylenediaminetetraacetic acid (EDTA) tubes: (4) Sodium Heparin tubes; (5) Citrate tubes with 3.2% sodium citrate solution; and (6) Glass Blood Collection tubes with acid citrate dextrose. Samples were tested against three different influenza viruses (A/California/07/2009 (H1N1pdm09), A/Texas/50/2012 (H3N2), and B/Massachusetts/2/2012) for hemagglutination inhibition titer and virus neutralization titer via a microneutralization (MN) assay, and data compared to that obtained for standard serum sample collected in SST.Results: HAI and MN titers against type A viruses were within two dilutions compared to SST collection method over 96% of the time irrespective of sample type or anticoagulant. However, HAI titers for type B virus were more variable across different collection methods. EDTA plasma samples were greater than two dilutions higher than SST serum samples 70% (21 of 30 samples) of the time. In contrast, MN titers were within two dilutions over 96% of the time, with the highest deviation noted in acid citrate dextrose plasma samples (3 of 30 samples tested, 10%).Conclusions: These data provide useful guidelines for sample collection and serology testing when screening: (i) influenza vaccine immunogenicity antibody response; (ii) antibody responses to newly emerging viral strains; and (iii) clinical samples for anti-influenza antibody activity. [ABSTRACT FROM AUTHOR]

A candidate vaccine (D1ME-VRP) expressing dengue virus type 1 premembrane and envelope proteins in a Venezuelan equine encephalitis (VEE) virus replicon particle (VRP) system was constructed and tested in conjunction with a plasmid DNA vaccine (D1ME-DNA) expressing identical dengue virus sequences. Cynomolgus macaques were vaccinated with three doses of DNA (DDD), three doses of VRP (VVV group), or a heterologous DNA prime-VRP boost regimen (DDV) using two doses of DNA vaccine and a third dose of VRP vaccine. Four weeks after the final immunization, the DDV group produced the highest dengue virus type 1-specific immunoglobulin G antibody responses and virus-neutralizing antibody titers. Moderate T-cell responses were demonstrated only in DDD- and DDV-vaccinated animals. When vaccinated animals were challenged with live virus, all vaccination regimens showed significant protection from viremia. DDV-immunized animals were completely protected from viremia (mean time of viremia = 0 days), whereas DDD- and VVV-vaccinated animals had mean times of viremia of 0.66 and 0.75 day, respectively, compared to 6.33 days for the control group of animals. [ABSTRACT FROM AUTHOR]