Enhanced Immunogenicity of Inactivated Dengue Vaccines by Novel Polysaccharide-Based Adjuvants in Mice.

Dengue fever, caused by any of four dengue viruses (DENV1-4), is a major global burden. Currently, there is no effective vaccine that prevents infection in dengue naïve populations. We tested the ability of two novel adjuvants (Advax-PEI and Advax-2), using aluminum hydroxide (alum) as control, to enhance the immunogenicity of formalin- or psoralen-inactivated (PIV or PsIV) DENV2 vaccines in mice. Mice were vaccinated on days 0 and 30, and serum samples were collected on days 30, 60, 90, and 101. Neutralizing antibodies were determined by microneutralization (MN) assays, and the geometric mean 50% MN (MN50) titers were calculated. For the PIV groups, after one dose MN50 titers were higher in the novel adjuvant groups compared to the alum control, while MN50 titers were comparable between the adjuvant groups after the second dose. For the PsIV groups, both novel adjuvants induced higher MN50 titers than the alum control after the second dose. Spleen cells were collected on days 45 and 101 for enzyme-linked immunospot (ELISPOT) for IFNγ and IL4. Both PIV and PsIV groups elicited different degrees of IFNγ and IL4 responses. Overall, Advax-2 gave the best responses just ahead of Advax-PEI. Given Advax-2's extensive human experience in other vaccine applications, it will be pursued for further development.

SARS-CoV-2 seropositivity and subsequent infection risk in healthy young adults: a prospective cohort study.

BACKGROUND: Whether young adults who are infected with SARS-CoV-2 are at risk of subsequent infection is uncertain. We investigated the risk of subsequent SARS-CoV-2 infection among young adults seropositive for a previous infection. METHODS: This analysis was performed as part of the prospective COVID-19 Health Action Response for Marines study (CHARM). CHARM included predominantly male US Marine recruits, aged 18-20 years, following a 2-week unsupervised quarantine at home. After the home quarantine period, upon arrival at a Marine-supervised 2-week quarantine facility (college campus or hotel), participants were enrolled and were assessed for baseline SARS-CoV-2 IgG seropositivity, defined as a dilution of 1:150 or more on receptor-binding domain and full-length spike protein ELISA. Participants also completed a questionnaire consisting of demographic information, risk factors, reporting of 14 specific COVID-19-related symptoms or any other unspecified symptom, and brief medical history. SARS-CoV-2 infection was assessed by PCR at weeks 0, 1, and 2 of quarantine and participants completed a follow-up questionnaire, which included questions about the same COVID-19-related symptoms since the last study visit. Participants were excluded at this stage if they had a positive PCR test during quarantine. Participants who had three negative swab PCR results during quarantine and a baseline serum serology test at the beginning of the supervised quarantine that identified them as seronegative or seropositive for SARS-CoV-2 then went on to basic training at Marine Corps Recruit Depot-Parris Island. Three PCR tests were done at weeks 2, 4, and 6 in both seropositive and seronegative groups, along with the follow-up symptom questionnaire and baseline neutralising antibody titres on all subsequently infected seropositive and selected seropositive uninfected participants (prospective study period). FINDINGS: Between May 11, 2020, and Nov 2, 2020, we enrolled 3249 participants, of whom 3168 (98%) continued into the 2-week quarantine period. 3076 (95%) participants, 2825 (92%) of whom were men, were then followed up during the prospective study period after quarantine for 6 weeks. Among 189 seropositive participants, 19 (10%) had at least one positive PCR test for SARS-CoV-2 during the 6-week follow-up (1·1 cases per person-year). In contrast, 1079 (48%) of 2247 seronegative participants tested positive (6·2 cases per person-year). The incidence rate ratio was 0·18 (95% CI 0·11-0·28; p<0·001). Among seropositive recruits, infection was more likely with lower baseline full-length spike protein IgG titres than in those with higher baseline full-length spike protein IgG titres (hazard ratio 0·45 [95% CI 0·32-0·65]; p<0·001). Infected seropositive participants had viral loads that were about 10-times lower than those of infected seronegative participants (ORF1ab gene cycle threshold difference 3·95 [95% CI 1·23-6·67]; p=0·004). Among seropositive participants, baseline neutralising titres were detected in 45 (83%) of 54 uninfected and in six (32%) of 19 infected participants during the 6 weeks of observation (ID50 difference p<0·0001). INTERPRETATION: Seropositive young adults had about one-fifth the risk of subsequent infection compared with seronegative individuals. Although antibodies induced by initial infection are largely protective, they do not guarantee effective SARS-CoV-2 neutralisation activity or immunity against subsequent infection. These findings might be relevant for optimisation of mass vaccination strategies. FUNDING: Defense Health Agency and Defense Advanced Research Projects Agency.

Flexible Zinc Oxide Nanowire Array/Graphene Nanohybrid for High-Sensitivity Strain Detection.

A fully flexible strain sensor consisting of vertically aligned ZnO nanowires on graphene transferred on polyethylene terephthalate with prefabricated Au/Ti electrodes (ZnO-VANWs/Gr)/PET) has been obtained. The ZnO-VANWs were grown in solution using a seedless hydrothermal process and are single-crystalline of (0001) orientation that provides optimal piezoelectric gating on graphene when deformed mechanically. The change of the graphene channel conductance under such a piezoelectric gating through transduction of the mechanical deformation on the ZnO-VANWs/Gr was used to detect the strain induced by the deformation. Under applied normal forces of 0.30, 0.50, and 0.70 N in a dynamic manner, the ZnO-VANWs/Gr/PET strain sensors exhibited a high response and response times of ∼0.20 s to both force on and off were achieved. Under mechanical bending curvatures of 0.18, 0.23, 0.37, and 0.45 cm-1, high sensitivity of the gauge factors up to ∼248 and response times of 0.20 s/0.20 s (rise/fall) were achieved on the ZnO-VANWs/Gr/PET strain sensors. Moreover, the response changes polarity when the directions of bending alters between up and down, corresponding to the polarity change of the space charge on the ZnO-VANWs/Gr interface as a consequence of the compressive and tensile strains along the ZnO-VANWs. This result shows that the low-cost and scalable ZnO-VANWs/Gr/PET strain sensors are promising for applications in stress/strain monitoring, wearable electronics, and touch screens.

Localized Surface Plasmon Resonance Enhanced Light Absorption in AuCu/CsPbCl3 Core/Shell Nanocrystals.

Localized surface plasmon resonance (LSPR) is shown to be effective in trapping light for enhanced light absorption and hence performance in photonic and optoelectronic devices. Implementation of LSPR in all-inorganic perovskite nanocrystals (PNCs) is particularly important considering their unique advantages in optoelectronics. Motivated by this, the first success in colloidal synthesis of AuCu/CsPbCl3 core/shell PNCs and observation of enhanced light absorption by the perovskite CsPbCl3 shell of thickness in the range of 2-4 nm, enabled by the LSPR AuCu core of an average diameter of 7.1 nm, is reported. This enhanced light absorption leads to a remarkably enhanced photoresponse in PNCs/graphene nanohybrid photodetectors using the AuCu/CsPbCl3 core/shell PNCs, by more than 30 times as compared to the counterparts with CsPbCl3 PNCs only (8-12 nm in dimension). This result illustrates the feasibility in implementation of LSPR light trapping directly in core/shell PNCs for high-performance optoelectronics.

Enhanced immunogenicity and protective efficacy of a tetravalent dengue DNA vaccine using electroporation and intradermal delivery.

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.

Controllable Synthesis of Monodispersed Fe1- xS2 Nanocrystals for High-Performance Optoelectronic Devices.

The optical properties of stoichiometric iron pyrite (FeS2) nanocrystals (NCs) are characterized by strong UV-Visible (UV-Vis) absorption within the cutoff while negligible absorption beyond the cutoff in near-infrared and longer wavelengths. Herein, we show this bandgap limitation can be broken through controllable synthesis of nonstoichiometric Fe1- xS2 NCs ( x = 0.01-0.107) to induce localized surface plasmonic resonance (LSPR) absorption beyond the cutoff to short-wave infrared spectrum (SWIR, 1-3 µm) with remarkably enhanced broadband absorption across UV-Vis-SWIR spectra. To illustrate the benefit of the broadband absorption, colloidal LSPR Fe1- xS2 NCs were printed on graphene to form LSPR Fe1- xS2 NCs/graphene heterostructure photodetectors. Extraordinary photoresponsivity in exceeding 4.32 × 106 A/W and figure-of-merit detectivity D* > 7.50 × 1012 Jones have been demonstrated in the broadband of UV-Vis-SWIR at room temperature. These Fe1- xS2 NCs/graphene heterostructures are printable and flexible and therefore promising for practical optical and optoelectronic applications.

High-Performance All-Inorganic CsPbCl3 Perovskite Nanocrystal Photodetectors with Superior Stability.

All-inorganic perovskites nanostructures, such as CsPbCl3 nanocrystals (NCs), are promising in many applications including light-emitting diodes, photovoltaics, and photodetectors. Despite the impressive performance that was demonstrated, a critical issue remains due to the instability of the perovskites in ambient. Herein, we report a method of passivating crystalline CsPbCl3 NC surfaces with 3-mercaptopropionic acid (MPA), and superior ambient stability is achieved. The printing of these colloidal NCs on the channel of graphene field-effect transistors (GFETs) on solid Si/SiO2 and flexible polyethylene terephthalate substrates was carried out to obtain CsPbCl3 NCs/GFET heterojunction photodetectors for flexible and visible-blind ultraviolet detection at wavelength below 400 nm. Besides ambient stability, the additional benefits of passivating surface charge trapping by the defects on CsPbCl3 NCs and facilitating high-efficiency charge transfer between the CsPbCl3 NCs and graphene were provided by MPA. Extraordinary optoelectronic performance was obtained on the CsPbCl3 NCs/graphene devices including a high ultraviolet responsivity exceeding 106 A/W, a high detectivity of 2 × 1013 Jones, a fast photoresponse time of 0.3 s, and ambient stability with less than 10% degradation of photoresponse after 2400 h. This result demonstrates the crucial importance of the perovskite NC surface passivation not only to the performance but also to the stability of the perovskite optoelectronic devices.

Inkjet-Printed Imbedded Graphene Nanoplatelet/Zinc Oxide Bulk Heterojunctions Nanocomposite Films for Ultraviolet Photodetection.

A ZnO sol-gel precursor (ZnOPr) and graphene nanoplatelets (GnPs) are mixed into a composite ink for inkjet printing photodetectors with bulk heterojunctions of ZnO/GnP on a heated SiO2/Si substrate. Heating of the SiO2/Si wafers at ∼50 °C was found optimal to prevent segregated droplets on the hydrophobic surface of the SiO2/Si substrate during printing. After printing the ZnO/GnP channels, thermal annealing at 350 °C for 2 h was performed for crystallization of ZnO and formation of the ZnO/GnP heterojunctions. The GnP concentration was varied from 0, 5, 20, and 30 mM to evaluate optimal formation of the ZnO/GnP bulk heterojunction nanocomposites based on ultraviolet photoresponse performance. The best performance was observed at the 20 mM GnP concentration with the photoresponsivity reaching 2.2 A/W at an incident ultraviolet power of 2.2 µW and a 5 V bias. This photoresponsivity is an order of magnitude better than the previously reported counterparts, including 0.13 mA/W for dropcasted ZnO-graphite composites and much higher than 0.5 A/W for aerosol printed ZnO. The improved performance is attributed to the ZnO/GnP bulk heterojunctions with improved interfaces that enable efficient exciton dissociation and the charge transport. The developed inkjet printing of sol-gel composite inks approach can be scalable and low cost for practical applications.

Influence of sample collection tube method, anticoagulant-containing plasma versus serum, on influenza virus hemagglutination inhibition titer and microneutralization titer serological assays.

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.

Heat-Assisted Inkjet Printing of Tungsten Oxide for High-Performance Ultraviolet Photodetectors.

An ammonium metatungstate precursor (WO3Pr) ink was printed for tungsten oxide (WO3) UV detectors on SiO2/Si wafers with prefabricated Au electrodes. A systematic study was carried out on the printing parameters including substrate temperatures in the range of 22-80 °C, WO3Pr molar concentrations of 0.01, 0.02, and 0.03 M, and printing scan numbers up to 7 to understand their effects on the resulted WO3 film morphology and optoelectronic properties. It has been found that the printing parameters can sensitively affect the WO3 film morphology, which in turn impacts the WO3 photodetector performance. In particular, the printed films experienced a systematic change from discontinuous droplets at below 40 °C to continuous films at 40-60 °C of the substrate temperature. At higher temperatures, the excessive heat from the substrate not only caused drastic evaporation of the printed ink, resulting in highly nonuniform films, but also detrimental heating of the ink in the printer nozzle in proximity of the substrate, preventing continuous printing operation. An optimal printing window of the substrate temperature of 45-55 °C at a molar concentration of 0.02 M of ammonium metatungstate and three printing scans was obtained for the best UV detector performance. A large on/off ratio of 3538 and a high responsivity up to 2.70 A/W at 5 V bias (0.54 A/W·V) represent a significant improvement over the best report of ∼0.28 µA/W·V on WOX photodetectors, which indicates that the printed WO3 films are promising for various applications of optoelectronics and sensors.

Printable Nanocomposite FeS2-PbS Nanocrystals/Graphene Heterojunction Photodetectors for Broadband Photodetection.

Colloidal nanocrystals are attractive materials for optoelectronics applications because they offer a compelling combination of low-cost solution processing, printability, and spectral tunability through the quantum dot size effect. Here we explore a novel nanocomposite photosensitizer consisting of colloidal nanocrystals of FeS2 and PbS with complementary optical and microstructural properties for broadband photodetection. Using a newly developed ligand exchange to achieve high-efficiency charge transfer across the nanocomposite FeS2-PbS sensitizer and graphene on the FeS2-PbS/graphene photoconductors, an extraordinary photoresponsivity in exceeding ∼106 A/W was obtained in an ultrabroad spectrum of ultraviolet (UV)-visible-near-infrared (NIR). This is in contrast to the nearly 3 orders of magnitude reduction of the photoresponsivity from ∼106 A/W at UV to 103 A/W at NIR on their counterpart of FeS2/graphene detectors. This illustrates the combined advantages of the nanocomposite sensitizers and the high charge mobility in FeS2-PbS/graphene van der Waals heterostructures for nanohybrid optoelectronics with high performance, low cost, and scalability for commercialization.

Quantum Dots-Facilitated Printing of ZnO Nanostructure Photodetectors with Improved Performance.

A nanocomposite ink composed of zinc oxide precursor (ZnOPr) and crystalline ZnO quantum dots (ZnOPrQDs) has been explored for printing high-performance ultraviolet (UV) photodetectors. The performance of the devices has been compared with their counterparts' printed from ZnOPr ink without ZnO QDs. Remarkably, higher UV photoresponsivity of 383.6 A/W and the on/off ratio of 2470 are observed in the former, which are significantly better than 14.7 A/W and 949 in the latter. The improved performance is attributed to the increased viscosity in the nanocomposite ink to enable a nanoporous structure with improved crystallinity and surface-to-volume ratio. This is key to enhanced surface electron-depletion effect for higher UV responsivity and on/off ratio. In addition, the QD-assisted printing provides a simple and robust method for printing high-performance optoelectronics and sensors.

Printable Transfer-Free and Wafer-Size MoS2/Graphene van der Waals Heterostructures for High-Performance Photodetection.

Two-dimensional (2D) MoS2/graphene van der Waals heterostructures integrate the superior light-solid interaction in MoS2 and charge mobility in graphene for high-performance optoelectronic devices. Key to the device performance lies in a clean MoS2/graphene interface to facilitate efficient transfer of photogenerated charges. Here, we report a printable and transfer-free process for fabrication of wafer-size MoS2/graphene van der Waals heterostructures obtained using a metal-free-grown graphene, followed by low-temperature growth of MoS2 from the printed thin film of ammonium thiomolybdate on graphene. The photodetectors based on the transfer-free MoS2/graphene heterostructures exhibit extraordinary short photoresponse rise/decay times of 20/30 ms, which are significantly faster than those of the previously reported MoS2/transferred-graphene photodetectors (0.28-1.5 s). In addition, a high photoresponsivity of up to 835 mA/W was observed in the visible spectrum on such transfer-free MoS2/graphene heterostructures, which is much higher than that of the reported photodetectors based on the exfoliated layered MoS2 (0.42 mA/W), the graphene (6.1 mA/W), and transfer-free MoS2/graphene/SiC heterostructures (∼40 mA/W). The enhanced performance is attributed to the clean interface on the transfer-free MoS2/graphene heterostructures. This printable and transfer-free process paves the way for large-scale commercial applications of the emerging 2D heterostructures in optoelectronics and sensors.

All-Printable ZnO Quantum Dots/Graphene van der Waals Heterostructures for Ultrasensitive Detection of Ultraviolet Light.

In ZnO quantum dot/graphene heterojunction photodetectors, fabricated by printing quantum dots (QDs) directly on the graphene field-effect transistor (GFET) channel, the combination of the strong quantum confinement in ZnO QDs and the high charge mobility in graphene allows extraordinary quantum efficiency (or photoconductive gain) in visible-blind ultraviolet (UV) detection. Key to the high performance is a clean van der Waals interface to facilitate an efficient charge transfer from ZnO QDs to graphene upon UV illumination. Here, we report a robust ZnO QD surface activation process and demonstrate that a transition from zero to extraordinarily high photoresponsivity of 9.9 × 108 A/W and a photoconductive gain of 3.6 × 109 can be obtained in ZnO QDs/GFET heterojunction photodetectors, as the ZnO QDs surface is systematically engineered using this process. The high figure-of-merit UV detectivity D* in exceeding 1 × 1014 Jones represents more than 1 order of magnitude improvement over the best reported previously on ZnO nanostructure-based UV detectors. This result not only sheds light on the critical role of the van der Waals interface in affecting the optoelectronic process in ZnO QDs/GFET heterojunction photodetectors but also demonstrates the viability of printing quantum devices of high performance and low cost.

A tetravalent dengue vaccine based on a complex adenovirus vector provides significant protection in rhesus monkeys against all four serotypes of dengue virus.

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.

A heterologous DNA prime-Venezuelan equine encephalitis virus replicon particle boost dengue vaccine regimen affords complete protection from virus challenge in cynomolgus macaques.

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.

A chimeric tetravalent dengue DNA vaccine elicits neutralizing antibody to all four virus serotypes in rhesus macaques.

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.

Needle-free Biojector injection of a dengue virus type 1 DNA vaccine with human immunostimulatory sequences and the GM-CSF gene increases immunogenicity and protection from virus challenge in Aotus monkeys.

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.