CpG-containing oligodeoxynucleotides augment and switch the immune responses of cattle to bovine herpesvirus-1 glycoprotein D

The adjuvanticity of a synthetic oligodeoxynucleotide containing unmethylated CpG motifs (CpG ODN) was determined in cattle. Calves were immunized with a truncated secreted version of glycoprotein D (tgD) of bovine herpes virus-1 (BHV-1) formulated with alum, CpG ODN, or a combination of both. BHV-1 tgD formulated with CpG ODN or with alum and CpG ODN induced a stronger and more balanced immune response than tgD in alum. This level of immunity was of sufficient magnitude to minimize weight loss and significantly reduce the duration of virus shedding after intranasal viral challenge. Local tissue reactions generated by CpG ODN were very mild and transient, whereas reactions induced by alum or a combination of CpG ODN and alum were moderate in severity and duration. These data demonstrate that CpG ODN causes minimal injection site reactions and yet acts as an effective adjuvant in cattle.     

Safety and efficacy of CpG-containing oligodeoxynucleotides as immunological adjuvants in rabbits

The efficacy of oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN) as an adjuvant for rabbits was assessed alone and in combination with aluminum hydroxide (CpG/alum). The CpG/alum combination elicited a greater immune response to several antigens compared to Freund's adjuvant. A non-CpG/alum combination did not have the same effects as CpG/alum suggesting that the adjuvanticity was related to the CpG motifs. In addition, we formulated one of the antigens with combinations of CpG ODN and 30 or 10% Emulsigen (Em) [CpG/Em (30%) and CpG/Em (10%)]. Both CpG/Em (30%) and CpG/Em (10%) were more effective than Em, and equivalent to CpG/alum. The CpG/Em (10%) combination caused minimal tissue damage. Our results demonstrate that the addition of CpG ODN to aluminum hydroxide or to 10% Em significantly improves the efficiency of these adjuvants, without enhancing tissue reaction.     

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

Background

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

Methodology and principal findings

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

Conclusions

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

Evaluation of immune responses to a Plasmodium vivax CSP-based recombinant protein vaccine candidate in combination with second-generation adjuvants in mice

Plasmodium vivax is the major cause of malaria outside of sub-Saharan Africa and causes morbidity and results in significant economic impact in developing countries. In order to produce a P. vivax vaccine for global use, we have previously reported the development of VMP001, based on the circumsporozoite protein (CSP) of P. vivax. Our interest is to evaluate second-generation vaccine formulations to identify novel combinations of adjuvants capable of inducing strong, long-lasting immune responses. In this study, groups of C57BL/6J mice were immunized subcutaneously three times with VMP001 emulsified with synthetic TLR4 (GLA) or TLR7/8 (R848) agonist in stable emulsion (SE), a combination of the TLR4 and TLR7/8 agonists, or SE alone. Sera and splenocytes were tested for the presence of antigen-specific humoral and cellular responses, respectively. All groups of mice generated high titers of anti-P. vivax IgG antibodies as detected by ELISA and immunofluorescence assay. GLA-SE promoted a shift in the antibody response to a Th1 profile, as demonstrated by the change in IgG2c/IgG1 ratio. In addition, GLA-SE induced a strong cellular immune response characterized by multi-functional, antigen-specific CD4(+) T cells secreting IL-2, TNF and IFN-γ. In contrast, mice immunized with SE or R848-SE produced low numbers of antigen-specific CD4(+) T cells, and these T cells secreted IL-2 and TNF, but not IFN-γ. Finally, R848-SE did not enhance the immune response compared to GLA-SE alone. Based on these results, we conclude that the combination of VMP001 and GLA-SE is highly immunogenic in mice and may serve as a potential second-generation vaccine candidate against vivax malaria.     

CpG oligodeoxynucleotides containing GACGTT motifs enhance the immune responses elicited by a goose parvovirus vaccine in ducks

Recombinant parvovirus VP2 (rVP2) was formulated with different types of adjuvant, including aluminum adjuvant and CpG oligodeoxynucleotides (ODNs), and the immunological responses after vaccination in ducks were examined. In comparison with the control group, production of rVP2-specific antibodies, expression of cytokines in peripheral blood mononuclear cells (PBMC) stimulated by rVP2, and percentage of CD4(+)/CD8(+) cells in PBMC were significantly increased in ducks immunized with rVP2 formulated with CpG ODNs containing 3 copies of GACGTT motif. CpG ODNs with GACGTT motifs might be used to improve the efficacy of vaccines for ducks.     

Analysis of the ability of five adjuvants to enhance immune responses to a chimeric plant virus displaying an HIV-1 peptide

The ability of five different adjuvants (alum, complete Freund's adjuvant, Quil A, AdjuPrime and Ribi) to stimulate humoral and T-cell mediated immune responses against a purified chimeric virus particle was investigated. Each adjuvant was administered subcutaneously to adult mice together with 10 microg of wildtype (wt) cowpea mosaic virus (CPMV) or a chimeric CPMV displaying the HIV-1 gp41 peptide, residues 731-752. All preparations elicited strong antibody responses to CPMV, but Quil A elicited the highest and most consistent responses to the HIV-1 peptide. This finding was reflected in both ELISA titres with immobilized peptide and in HIV-1-neutralizing antibody. In addition Quil A was also, the only adjuvant to stimulate an in vitro proliferative T-cell response. Surprisingly with all adjuvant formulations a predominately IgG2a anti-gp41 peptide response was observed, indicating a type 1 T-helper cell-like response. Furthermore, the efficiency of the CPMV display system was demonstrated by its ability to induce good levels of peptide specific antibody in the absence of any adjuvant.     

Use of a polyanionic carbomer, Carbopol971P, in combination with MF59, improves antibody responses to HIV-1 envelope glycoprotein

Identification of optimal antigen(s) and adjuvant combination(s) to elicit potent, protective, and long-lasting immunity has been a major challenge for the development of effective vaccines against chronic viral pathogens, such as HIV-1, for which there are not yet any licensed vaccines. Here we describe the use of a novel adjuvant approach employing Carbopol 971P^® NF (hereafter referred to as Carbopol971P), a cross-linked polyanionic carbomer, in combination with the Novartis proprietary oil-in-water adjuvant, MF59, as a potentially safe and effective adjuvant to augment humoral immune responses to the HIV-1 envelope glycoprotein (Env). Intramuscular immunization of small animals with recombinant Env glycoprotein (gp140) formulated in Carbopol971P plus MF59 gave significantly higher titers of binding and virus neutralizing antibodies as compared to immunization using gp140 with either MF59 or Carbopol971P alone. In addition, the antibodies generated were of higher avidity. Importantly, the use of Carbopol971P plus MF59 did not cause any serious adverse reactions or any obvious health problems in animals upon intramuscular administration. Hence, the Carbopol971P plus MF59 adjuvant formulation may provide a benefit for future vaccine applications.     

Immune responses in bovines to recombinant glycoprotein D of bovine herpesvirus type 5 as vaccine antigen

Bovine herpesvirus 5 (BoHV-5) is responsible for outbreaks of meningoencephalitis that cause important economic losses in young cattle. BoHV-5 glycoprotein D (gD5) is essential for attachment and penetration into permissive cells and targeting of host immune systems, inducing strong humoral and cellular immune responses. The aim of this study was to evaluate the vaccinal immune response of vaccines formulated with the recombinant BoHV-5 gD (rgD5) in bovines. For the experiment, 72 heifers were randomly allotted into 6 different groups with 12 animals each. Group 1: vaccine formulated using inactivated BoHV-5 (iBoHV-5) adjuvanted with ISA50V2; Group 2: iBoHV-5 associated with 100 µg of rgD5 adjuvanted with ISA50V2; Group 3: 100 µg of rgD5 adjuvanted with ISA50V2; Group 4: 100 µg of rgD5 adjuvanted with Al(OH)₃; Group 5: commercial vaccine; and Group 6: control group. Two doses were administered in a 26-day interval and the third after 357 days from primo vaccination. Cattle vaccinated with the vaccines formulated with iBoHV-5 plus rgD5 showed a significant (p < 0.01) five-fold increase in total immunoglobulin G (IgG) for BoHV-5, BoHV-1, and rgD5 as compared with the commercial and control groups. Also, a significant (p < 0.05) increase in IgG1 and IgG2a levels was induced in serum for rgD5. In addition, these same vaccines showed significant (p < 0.01) four-fold higher titers of BoHV-1 and -5 neutralizing antibodies. The results demonstrated that the rgD5 conserved important epitopes that were able to stimulate bovine humoral immunity response capable of viral neutralization of BoHV-1 and -5, suggesting it as a promising vaccine antigen to be used in vaccine for BoHV-1 and -5 endemic areas.     

Antibody persistence, PRP-specific immune memory, and booster responses in infants immunised with a combination DTPa-HBV-IPV/Hib vaccine

A new single-injection combination vaccine against six diseases has been developed to accommodate the growing number of recommended paediatric vaccines. A pentavalent liquid diphtheria, tetanus, acellular pertussis (3-component), hepatitis B, and inactivated polio (types 1-3) combined vaccine (DTPa-HBV-IPV) is extemporaneously mixed with a lyophilized Haemophilus influenza type B (Hib) conjugate vaccine (polyribosyl-ribitol phosphate (PRP)-T) and given as a single-injection. A cohort of 368 healthy infants was initially studied to evaluate the immunogenicity and reactogenicity of this hexavalent combination given as a primary course at 2, 4, and 6 months of age. At 15 months of age, from this cohort, 219 children received a booster dose of a licensed DTPa/Hib (PRP-T) vaccine to assess the booster response, while 70 received a challenge dose of unconjugated PRP (PRP) vaccine (to evaluate Hib-specific memory) plus a separate DTPa vaccine. Seven to 10 days following plain PRP challenge, anti-PRP geometric mean antibody concentrations (GMCs) had increased 13-fold to 5.67 microg/ml, and thirty days after conjugated PRP booster vaccination, anti-PRP antibody GMCs increased 102-fold. Both responses are indicative of immune memory. Vaccination was well tolerated following all primary and booster doses, although 10.5% of booster recipients experienced >50-mm local swelling at the site of DTPa vaccination. We conclude that DTPa-HBV-IPV/Hib is safe and immunogenic for primary vaccination, and that Hib-specific memory is induced by primary vaccination.     

Methylglycol chitosan and a synthetic TLR4 agonist enhance immune responses to influenza vaccine administered sublingually

Influenza is a vaccine-preventable contagious respiratory illness caused by influenza (flu) viruses which can lead to hospitalization and sometimes even death. Current flu vaccines delivered intramuscularly (IM) or intradermally (ID) are less effective at eliciting protective mucosal immune responses and vaccines delivered intranasally (IN) possess potential safety concerns. Sublingual (SL) vaccination is a promising alternative route for vaccine delivery which has been indicated as safe and effective at inducing protective immune responses in both systemic and mucosal compartments. We evaluated the efficacy of methylglycol chitosan (MGC) and a synthetic toll-like receptor 4 agonist (CRX-601), alone or in combination, for improving systemic and mucosal immune responses to a monovalent detergent-split flu virus vaccine delivered SL. SL vaccination of mice with split-flu vaccine formulated with either MGC or CRX-601 resulted in specific serum IgG and mucosal IgA titers that were significantly greater than titers from non-adjuvanted vaccination and equivalent to or greater than titers in mice vaccinated IM. Our results demonstrate that SL vaccination utilizing MGC or CRX-601 as adjuvants is a viable alternative route of vaccination for flu which can elicit systemic immune responses equivalent to or greater than IM vaccination with the added benefit of stimulating a robust specific mucosal immune response.     

Nanoparticle size influences the magnitude and quality of mucosal immune responses after intranasal immunization

Background

The development of nanoparticulate antigen-delivery systems is an important emerging area of vaccinology, being sought to amplify immune responses to recombinant antigens that are poorly immunogenic. Nanoparticle size may play an important role in influencing the activity of such particulate-based adjuvants.

Methods

To explore how the size of nanoparticles that are in the range of many common viruses can modulate the magnitude and quality of mucosal immune responses, the model antigen ovalbumin (OVA) was conjugated to 30 nm or 200 nm polypropylene sulfide nanoparticles (NPs) and administered intranasally to C57BL/6 mice.

Results

We show that by increasing the size of the NPs from 30 to 200 nm, OVA was more effectively delivered into both MHC class I and MHC class II-presentation pathways. Intranasal immunization with the 200 nm NPs increased the magnitude of CD4⁺ T cell responses in the lungs, as well as systemic and mucosal humoral responses. Most importantly, 200 nm NPs increased the proportion of antigen-specific polyfunctional CD4⁺ T cells as compared to 30 nm NPs.

Conclusions

The 200 nm NPs are a very interesting antigen nanocarrier for prophylactic vaccines against mucosal pathogens that require multifunctional CD4⁺ T cells for protection. These results contribute to our understanding of how the size of an antigen-conjugated nanoparticle modulates mucosal immune responses to a protein antigen and may be useful to engineer subunit vaccines able to elicit appropriate mucosal immune responses that correlate with protection.     

Adjuvant activity of Quillaja brasiliensis saponins on the immune responses to bovine herpesvirus type 1 in mice

The chemical characterization of aqueous extracts (AE) of barks, leaves and branches and the saponin fraction denominated QB-90 obtained from Quillaja brasiliensis, a native species from Southern Brazil, show remarkable similarities to Quillaja saponaria saponins which are known as adjuvants in vaccine formulations.In vivo toxicity assays of AE and QB-90 showed not to be lethal for mice in doses ranging from 50 to 1600 microg and 50-400 microg, respectively. Experimental vaccines prepared with bovine herpesvirus type 1 (BHV-1) antigen and either AE (barks 100 microg, leaves 400 microg, branches 400 microg) or QB-90 (100 microg) were able to enhance the immune responses of mice in a comparable manner to saponins from Q. saponaria (QuilA, 100 microg). BHV-1 specific IgG, IgG1 and IgG2a antibody levels in serum were also significantly enhanced by AE, QB-90 and QuilA compared to control group (p<0.05). These results showed that AE and QB-90 from Q. brasiliensis are potential candidates as adjuvants in vaccines.     

Comparison of immune responses and protective efficacy of suicidal DNA vaccine and conventional DNA vaccine encoding glycoprotein C of pseudorabies virus in mice

In the present study, the immunogenicity and protective efficacy of a suicidal DNA vaccine (pSFVC1.5) incorporating Semliki Forest virus (SFV) replicon and expressing glycoprotein C (gC) of pseudorabies virus (PrV) was investigated and compared with a conventional plasmid DNA vaccine (pcDC) encoding the same antigen. In vitro, pSFVC1.5 could express gC protein and induce apoptosis of the transfected cells. After immunization in BALB/c mice, the gC-specific ELISA antibodies and neutralizing antibodies induced by pSFVC1.5 were relatively lower than those obtained in mice immunized with pcDC. However, mice immunized with pSFVC1.5 could confer more efficient protection than pcDC (100 and 62.5%, respectively) when challenged with the field PrV at 4 weeks after secondary immunization. Further analyses of cell-mediated immune responses showed that pSFVC1.5 induced stronger lymphocyte proliferative responses and higher levels of IFN-gamma, suggesting pSFVC1.5 could induce an enhanced Th1-type immune response. Collectively these results indicated that suicidal DNA vaccine is an alternative strategy to conventional DNA vaccine and can be considered a promising approach for the development of an efficacious vaccine against PrV.     

Cell-mediated immune responses to a varicella-zoster virus glycoprotein E vaccine using both a TLR agonist and QS21 in mice

Lack of adequate cell-mediated immunity (CMI) to varicella-zoster virus (VZV) has been associated with higher risks of developing herpes zoster (HZ) and associated post-herpetic neuralgia (PHN), and is of particular concern for older and immunocompromised individuals. Thus, the development of an effective HZ vaccine with a clinically acceptable safety profile that is capable of addressing decreased immunity would be highly desirable. In this study we compared the immunogenicity of different vaccine formulations containing VZV glycoprotein E (gE), an important target for CMI and antibody responses, in a VZV-primed mouse model. The formulations included recombinant gE, either unadjuvanted, or combined with aluminium salt or an Adjuvant System (AS01 or AS02), and CMI was used as the primary immunological endpoint. All adjuvanted vaccines induced gE- and/or VZV-specific CD4(+) T cell and antibody responses. A formulation of gE with an Adjuvant System containing the immunostimulants QS21 and 3-O-desacyl-4'-monophosphoryl lipid A (MPL) was shown to be more immunogenic than gE with aluminium salt or unadjuvanted gE (gE/saline). Both immunostimulants were shown to act synergistically in enhancing CMI responses. Formulations with AS01 elicited high frequencies of CD4(+) T cells producing IFN-γ and IL-2. These responses were dose-dependent with respect to both antigen and adjuvant. The gE/AS01(B) candidate vaccine induced higher frequencies of CD4(+) T cells producing IL-2 and/or IFN-γ than all other gE/AS01 formulations, supporting its use for clinical evaluations.     

Co-administration of polyphosphazenes with CpG oligodeoxynucleotides strongly enhances immune responses in mice immunized with Hepatitis B virus surface antigen

An emerging paradigm in vaccinology is that multiple adjuvant combinations may be more effective than individual adjuvants in enhancing immune responses to vaccine antigens. We investigated whether the polyphosphazenes used in combination with CpG oligodeoxynucleotides (ODN) were potent adjuvant formulations. BALB/c mice were immunized subcutaneously with Hepatitis B surface antigen (HBsAg) alone, or in various combinations with poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP), poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) or CpG ODN. All three adjuvants enhanced HBsAg-specific IgG1 antibody responses with PCEP inducing the highest responses. PCEP and CpG ODN significantly enhanced the Th1-associated antibody isotype IgG2a. As expected CpG ODN induced predominantly Th1-type immune responses while PCEP was associated with mixed Th1/Th2 immune responses. Interestingly, PCEP and PCPP synergized with CpG ODN to further enhance antibody responses. Since the mechanisms which mediate the adjuvant activity of polyphosphazenes are not fully understood, we investigated whether PCEP and PCPP could stimulate innate immune responses. Incubation of mouse splenocytes with PCEP or PCPP (in the absence of antigen) stimulated production of IL-4 and IL-12, but only PCEP induced significant IFNγ production. Additionally, IL-12 was not required for PCEP induced IFNγ response. We conclude that the polyphosphazene–CpG ODN combination is a potent adjuvant formulation that is more effective in enhancing immune responses than either of the individual adjuvants. In addition, we provide evidence that PCEP and PCPP can stimulate innate cytokine production, suggesting a potential mechanism by which polyphosphazenes achieve their potent adjuvant effects.     

Augmentation of immune responses to SARS coronavirus by a combination of DNA and whole killed virus vaccines

We studied the immunogenicity of a DNA SARS-vaccine, a whole killed virus, or a whole killed and DNA vaccine combination. The DNA vaccine contained a plasmid encoding the SARS coronavirus (SARS-CoV) S protein under the control of the human CMV promoter and intron A. The whole killed virus vaccine was comprised of SARS-CoV, propagated in Vero-E6 cells, with subsequent beta-propilactone inactivation and formulated with aluminum hydroxide adjuvant. Mice immunized twice with the DNA vaccine and once with the whole killed virus elicited higher antibody responses than mice immunized three times with the DNA vaccine or once with the whole killed virus vaccine. Mice immunized twice with the whole killed virus vaccine elicited higher antibody responses than mice immunized three times with the DNA vaccine or once with the whole killed virus vaccine. However, a combination of the vaccines induced T-helper type 1 (Th1) immune responses while the whole killed virus vaccine induced T helper type 2 (Th2) immune response. These results demonstrate that combination of the DNA vaccine and the whole killed virus vaccine can be used to enhance the magnitude and change the bias of the immune responses to SARS-CoV.     

Comparison of a glycoprotein E-based ELISA with a varicella-zoster whole-virus ELISA for the quantification of varicella vaccine immune responses in young children

Antibody response against varicella-zoster virus (VZV) is frequently assessed by whole-virus- (anti-VZV) or glycoprotein-based ELISAs. This study compared antibody concentrations measured by an assay quantifying anti-VZV glycoprotein E (anti-gE) and anti-VZV ELISA in 12–23-month-olds, receiving two varicella vaccine doses in a phase III trial (NCT02570126). Samples (pre- and 42 days post-each vaccination) initially tested with anti-VZV ELISA were re-tested with anti-gE ELISA. Of 1138 samples from 397 children, 757 were positive by anti-VZV (antibody concentration ≥25 mIU/mL) and 758 by anti-gE ELISA (≥97 mIU/mL). There were 375 double-negative and only 11 discrepant samples. The overall agreement was 99.03% (95% confidence interval: 98.28–99.52; McNemar p-value = 1). The ratio between antibody geometric mean concentrations (anti-gE/anti-VZV) for the 752 double-positive samples was 3.78 overall, 4.75 post-first, and 3.01 post-second vaccination. The anti-gE ELISA is a valid alternative for trials assessing antibody response to new varicella vaccines versus established ones, used as control.     

Integration of needle-free jet injection with advanced electroporation delivery enhances the magnitude,kinetics, and persistence of engineered DNA vaccine induced immune responses

The combination of optimized DNA constructs, improved formulations and advanced in vivo electroporation (EP) has been shown to generate potent and efficacious immune responses in the clinic. Needle-free jet injection has also been reported to improve DNA vaccine delivery over standard needle and syringe in clinical trials. Here we investigated the impact of combined jet injection and EP (Jet-EP) delivery on muscle transfection efficiency and DNA vaccine immunogenicity in rabbits and nonhuman primates (NHPs) compared to jet injection alone. Our results show that the addition of EP significantly enhanced in vivo DNA transfection efficiency of rabbit muscle over jet injection alone. Jet-EP delivery augmented the rate and magnitude of DNA vaccine induced humoral and cellular responses over jet injection alone in both rabbits and NHPs. Jet-EP delivery also resulted in higher proportions of polyfunctional antigen specific T cells producing IFNγ, IL-2, and/or TNFα. Elevated antibody levels were sustained nine months post immunization in NHPs immunized with a DNA vaccine using Jet-EP delivery, far outperforming jet delivery alone. Our results provide proof-of-concept that addition of advanced EP to needle-free jet injection delivery improves in vivo DNA transfection efficiency, increasing the magnitude, rate and duration of cellular and humoral immune responses to DNA vaccines. This combination likely has significant advantages in important vaccine and immunotherapy settings.     

Modulation of antigen-specific cellular immune responses to DNA vaccination in rhesus macaques through the use of IL-2, IFN-gamma, or IL-4 gene adjuvants

Extensive experiments have shown DNA vaccines' ability to elicit immune responses in vivo in a safe and well-tolerated manner in several model systems, including rodents and non-human primates. As the DNA-based vaccine and immunotherapy approaches are being explored in humans, significant efforts have also been focused on further improving the immune potency of this technology. One strategy to enhance immune responses for DNA vaccines is the use of molecular or genetic adjuvants. These molecular adjuvant constructs (which encodes for immunologically important molecules such as cytokines) can be co-administered along with DNA vaccine constructs. Once delivered, these adjuvants have shown to modulate the magnitude and direction (humoral or cellular) of the vaccine-induced immune responses in rodent models. To date, however, there has been very little data reported from studies in primates. In this study, we examined the effects of cytokine gene adjuvants to enhance the level of cell-mediated immune responses in rhesus macaques. We co-immunized rhesus macaques with expression plasmids encoding for IL-2, IFN-gamma or IL-4 cytokines along with the DNA vaccine constructs encoding for HIV env/rev (pCEnv) and SIV gag/pol (pCSGag/pol) proteins. We observed that coadministration of IL-2 and IFN-gamma cDNA resulted in enhancement of antigen-specific T cell-mediated immune responses.