Zika Virus Envelope Domain III Recombinant Protein Delivered With Saponin-Based Nanoadjuvant From Quillaja brasiliensis Enhances Anti-Zika Immune Responses, Including Neutralizing Antibodies and Splenocyte Proliferation.

Nanoadjuvants that combine immunostimulatory properties and delivery systems reportedly bestow major improvements on the efficacy of recombinant, protein-based vaccines. Among these, self-assembled micellar formulations named ISCOMs (immune stimulating complexes) show a great ability to trigger powerful immunological responses against infectious pathogens. Here, a nanoadjuvant preparation, based on saponins from Quillaja brasiliensis, was evaluated together with an experimental Zika virus (ZIKV) vaccine (IQB80-zEDIII) and compared to an equivalent vaccine with alum as the standard adjuvant. The preparations were administered to mice in two doses (on days zero and 14) and immune responses were evaluated on day 28 post-priming. Serum levels of anti-Zika virus IgG, IgG1, IgG2b, IgG2c, IgG3 were significantly increased by the nanoadjuvant vaccine, compared to the mice that received the alum-adjuvanted vaccine or the unadjuvanted vaccine. In addition, a robust production of neutralizing antibodies and in vitro splenocyte proliferative responses were observed in mice immunized with IQB80-zEDIII nanoformulated vaccine. Therefore, the IQB80-zEDIII recombinant preparation seems to be a suitable candidate vaccine for ZIKV. Overall, this study identified saponin-based delivery systems as an adequate adjuvant for recombinant ZIKV vaccines and has important implications for recombinant protein-based vaccine formulations against other flaviviruses and possibly enveloped viruses.

Protective efficacy of a live attenuated Mycoplasma hyopneumoniae vaccine with an ISCOM-matrix adjuvant in pigs.

An attenuated Mycoplasma hyopneumoniae vaccine that requires intrathoracic administration is commercially available for use against mycoplasmal pneumonia in China. Given the limitations of such a route of administration, this study was undertaken to assess the capacity of an ISCOM-matrix adjuvant to enhance immunogenicity following intramuscular use. Immune responses in pigs following vaccination and subsequent intra-tracheal bacterial inoculation were examined using lymphocyte proliferation, serology and mucosal IgA in both nasal and saliva swabs. Vaccination induced clear lymphocyte proliferation, but only slight serum antibody responses although these were significantly increased following experimental infection. Mucosal IgA was not detected in either nasal or salivary secretions. Following bacterial challenge, animals vaccinated with the adjuvant-containing live vaccine exhibited less severe pulmonary lesions (median score 3.67) than unvaccinated pigs (median score 13.58). The degree of ciliary loss on the respiratory tract surface was reduced in vaccinated pigs compared with experimentally infected controls. The findings indicated that the adjuvant vaccine administered IM provided protection against experimentally induced mycoplasmal pneumonia and could have commercial potential.

An overview of adjuvant formulations and delivery systems.

Adjuvants may promote immune responses: by recruiting professional antigen-presenting cells (APCs) to the vaccination site, increasing the delivery of antigens to APCs, or by activating APCs to produce cytokines and by triggering T cell responses. Aluminium salts have been effective at promoting protective humoral immunity; however, they are not effective in generating cell-mediated immunity. A number of different approaches have been developed to potentiate immune response and they have been partially successful. Research has been conducted into vaccine delivery systems (VDS); enhancing cross-presentation by targeting antigens to (APCs). Antigen discovery has increased over the past decade, and consequently, it has accelerated vaccine development demanding a new generation of VDS that combines different types of adjuvants into specific formulations with greater activity. The new approaches offer a wide spectrum of opportunities in vaccine research with direct applications in the near future.

Coadministration of polyinosinic:polycytidylic acid and immunostimulatory complexes modifies antigen processing in dendritic cell subsets and enhances HIV gag-specific T cell immunity.

Currently approved adjuvants induce protective Ab responses but are more limited for generating cellular immunity. In this study, we assessed the effect of combining two adjuvants with distinct mechanisms of action on their ability to prime T cells: the TLR3 ligand, polyinosinic:polycytidylic acid (poly I:C), and immunostimulatory complexes (ISCOMs). Each adjuvant was administered alone or together with HIV Gag protein (Gag), and the magnitude, quality, and phenotype of Gag-specific T cell responses were assessed. For CD8 T cells, all adjuvants induced a comparable response magnitude, but combining poly I:C with ISCOMs induced a high frequency of CD127(+), IL-2-producing cells with decreased expression of Tbet compared with either adjuvant alone. For CD4 T cells, combining poly I:C and ISCOMs increased the frequency of multifunctional cells, producing IFN-γ, IL-2, and TNF, and the total magnitude of the response compared with either adjuvant alone. CD8 or CD4 T cell responses induced by both adjuvants mediated protection against Gag-expressing Listeria monocytogenes or vaccinia viral infections. Poly I:C and ISCOMs can alter Ag uptake and/or processing, and we therefore used fluorescently labeled HIV Gag and DQ-OVA to assess these mechanisms, respectively, in multiple dendritic cell subsets. Poly I:C promoted uptake and retention of Ag, whereas ISCOMs enhanced Ag degradation. Combining poly I:C and ISCOMs caused substantial death of dendritic cells but persistence of degraded Ag. These data illustrate how combining adjuvants, such as poly I:C and ISCOMs, that modulate Ag processing and have potent innate activity, can enhance the magnitude, quality, and phenotype of T cell immunity.

Phenotypic analysis of ovine antigen presenting cells loaded with nanoparticles migrating from the site of vaccination.

Virus-sized particulate adjuvants such as ISCOMs, polystyrene nanoparticles and virus-like particles have been shown to target dendritic cells, resulting in the activation of T and B cells in vivo. Using an ovine pseudo-afferent lymph cannulation model to capture APC that traffic from the site of injection to the local lymph node, we show that 40-50 nm nanoparticles are taken up at the site of injection by dendritic cells (DCs) migrating to the draining lymph node. These DCs can express CD11c, CD1b, CD5, MHC class II and CD8. Nanoparticles transported by DCs migrating from the site of injection to the local lymph node therefore needs to be considered as a new mechanism underlying the immunogenicity of virus-sized vaccine delivery systems.

Structural studies of an immunostimulating gluco-arabinan from seeds of Caesalpinia bonduc.

A water-soluble gluco-arabinan (PS-II, M(W)∼62 kDa) isolated from the alkaline extract of the endosperm of Caesalpinia bonduc showed the presence of T-Glcp, (1→4)-Glcp, (1→2,3)-Glcp, T-Araf, (1→5)-Araf, (1→2,5)-Araf, and (1→2,3,5)-Araf in a relative proportion of approximately 2:2:2:3:2:1:1. The proposed repeating unit of the polysaccharide possessed a branched backbone of two (1→3)-α-D-glucopyranose followed by four (1→5)-α-L-arabinofuranose residues. In case of two (1→3)-α-D-glucopyranose, branching occurs at O-2 by a same residue terminated by another one at O-4 position. Out of four (1→5)-α-l-arabinofuranose residues, one residue is terminated at O-2 and O-3 by two arabinofuranose residues and another one situated at the adjacent position is terminated at O-2 with same residue, and two (1→5)-α-L-arabinofuranose residues are free from branching and located before and after the two branched arabinofuranose residues. This gluco-arabinan molecule and previously reported arabinan showed similar extent of splenocytes and thymocytes stimulation, but arabinan showed appreciable macrophage activations.

Functionalization of immunostimulating complexes (ISCOMs) with lipid vinyl sulfones and their application in immunological techniques and therapy.

BACKGROUND: Immunostimulating complexes (ISCOM)-type nanocapsules have been functionalized with lipid vinyl sulfones that anchor to them via the hydrophobic zone of their structure and can be charged with pharmacologically active molecules or macromolecules. These functionalized nanocapsules can incorporate protein A and bind to G immunoglobulins (IgGs) to make vehicles directed at the surface antigens of infectious agents, tumor cells, or receptor cells and deliver the encapsulated molecules in a highly specific way. They may be of particular use in pharmacological treatments with highly toxic molecules that should not be used in solution whenever it can be avoided. When bound to antibodies they can be used in biological processes that require the delivery or presentation of macromolecules to certain specific cells, in immunization processes for instance, or in diagnostic immunological techniques, as they are able to transport both the secondary antibodies and the reaction labels. METHODS AND RESULTS: We describe the preparation of ISCOMs, the binding to the ISCOMS of newly synthesized compounds composed of chain alkyl vinyl sulfone, and the subsequent binding of the vinyl-sulfone compounds to IgGs. Within this context, a compound deriving from cholesterol functionalized with vinyl sulfone and used together with cholesterol in varying proportions has been linked to the structure of the ISCOMs and bound to protein A-IgG. This functionalization in no way altered the form or structure of the ISCOMs and allowed the nanocapsules carrying the specific IgGs to bind to forms of Trypanosoma cruzi against which antibodies had been developed. The fact that functionalized ISCOMs containing antibodies could deliver actinomycin D directly to the parasite meant that the effective dose of the antibiotic could be reduced very significantly. CONCLUSION: We have developed ISCOM-type nanocapsules functionalized with lipid vinyl sulfone capable of anchoring to the surface of functional IgGs, which favors the recognition and transport of these nanocapsules precisely to certain kinds of cell.

[Enhancing the immunogenic activity of influvac vaccine in the use of adjuvant TI complexes modified by echinochrome A].

The self-assembly of marine macrophyte glycolipids, holothurian saponin, and cholesterol gave rise to nanoscale morphological structures called tubular immunostimulating (TI) complexes. Whether the latter could be used on the basis of vaccine preparations containing the influenza virus subunit antigens was studied. There was an obvious increase in the immunogenicity of influenza virus hemagglutinin when the experimental animals were immunized with this antigen as part of TI complexes. It was shown that the adjuvant activity of the TI complex to influenza virus hemagglutinin could be enhanced by adding the known antioxidant echinochrome A from a sand-dollar (Echinarachnius parma) to the matrix of the TI complex.

Global transcriptional response to ISCOM-Matrix adjuvant at the site of administration and in the draining lymph node early after intramuscular injection in pigs.

ISCOM vaccines induce a balanced Th1/Th2 response, long-lasting antibody responses and cytotoxic T lymphocytes. The mode of action for the adjuvant component, the ISCOM-Matrix, is known to some extent but questions remain regarding its mechanism of action. The Affymetrix GeneChip® Porcine Genome Array was applied to study the global transcriptional response to ISCOM-Matrix in pigs at the injection site and in the draining lymph node 24h after i.m. injection. Gene enrichment analysis revealed inflammation, innate immunity and antigen processing to be central in the ISCOM-Matrix response. At the injection site, 594 genes were differentially expressed, including up-regulation of the cytokines osteopontin (SPP1), IL-10 and IL-18 and the chemokines CCL2, CCL19 and CXCL16. Of the 362 genes differentially expressed in the lymph node, IL-1ß and CXCL11 were up-regulated whereas IL18, CCL15 and CXCL12 were down-regulated. ISCOM-Matrix also modulated genes for pattern recognition receptors at the injection site (TLR2, TLR4, MRC1, PTX3, LGALS3) and in the lymph node (TLR4, RIG-I, MDA5, OAS1, EIF2AK2, LGALS3). A high proportion of up-regulated interferon-regulated genes indicated an interferon response. Thus, several genes, genetic pathways and biological processes were identified that are likely to shape the early immune response elicited by ISCOM-based vaccines.

Vaccination of healthy and diseased koalas (Phascolarctos cinereus) with a Chlamydia pecorum multi-subunit vaccine: evaluation of immunity and pathology.

Chlamydial infections represent a major threat to the long-term survival of the koala and a successful vaccine would provide a valuable management tool. Vaccination however has the potential to enhance inflammatory disease in animals exposed to a natural infection prior to vaccination, a finding in early human and primate trials of whole cell vaccines to prevent trachoma. In the present study, we vaccinated both healthy koalas as well as clinically diseased koalas with a multi-subunit vaccine consisting of Chlamydia pecorum MOMP and NrdB mixed with immune stimulating complex as adjuvant. Following vaccination, there was no increase in inflammatory pathological changes in animals previously infected with Chlamydia. Strong antibody (including neutralizing antibodies) and lymphocyte proliferation responses were recorded in all vaccinated koalas, both healthy and clinically diseased. Vaccine induced antibodies specific for both vaccine antigens were observed not only in plasma but also in ocular secretions. Our data shows that an experimental chlamydial vaccine is safe to use in previously infected koalas, in that it does not worsen infection-associated lesions. Furthermore, the prototype vaccine is effective, as demonstrated by strong levels of neutralizing antibody and lymphocyte proliferation responses in both healthy and clinically diseased koalas. Collectively, this work illustrates the feasibility of developing a safe and effective Chlamydia vaccine as a tool for management of disease in wild koalas.

Bovine respiratory syncytial virus ISCOMs-Immunity, protection and safety in young conventional calves.

Bovine respiratory syncytial virus (BRSV) is a major cause of bronchiolitis and pneumonia in cattle and causes yearly outbreaks with high morbidity in Europe. Commercial vaccines against this virus needs improvement of efficacy, especially in calves with BRSV-specific maternally derived antibodies (MDA). We previously reported that an experimental BRSV-ISCOM vaccine, but not a commercial vaccine, induced strong clinical and virological protection in calves with MDA, immunized at 7-15 weeks of age. The aim of the present study was to characterize the immune responses, as well as to investigate the efficacy and safety in younger animals, representing the target population for vaccination. Four groups of five 3-8 week old calves with variable levels of BRSV-specific MDA were immunized s.c. twice at a 3 weeks interval with (i) BRSV immunostimulating complexes (BRSV-ISCOMs), (ii) BRSV-protein, (iii) adjuvant, or (iv) PBS. All calves were challenged with virulent BRSV by aerosol 2 weeks later and euthanized on day 6 after infection. The cellular and humoral responses were monitored as well as the clinical signs, the viral excretion and the pathology following challenge. Despite presence of MDA at the time of the immunization, only a minimum of clinical signs were observed in the BRSV-ISCOM group after challenge. In contrast, in all control groups, clinical signs of disease were observed in most of the animals (respiratory rates up to 76min(-1) and rectal temperatures up to 41°C). The clinical protection was associated to a highly significant reduction of virus replication in the upper and lower respiratory tract of calves, rapid systemic and local antibody responses and T helper cell responses dominated by IFNγ production. Animals that did not shed virus detectable by PCR or cell culture following challenge possessed particularly high levels of pulmonary IgA. The protective immunological responses to BRSV proteins and the ability to overcome the inhibiting effect of MDA were dependent on ISCOM borne antigen presentation.

A novel non-toxic combined CTA1-DD and ISCOMS adjuvant vector for effective mucosal immunization against influenza virus.

Here we demonstrate that by using non-toxic fractions of saponin combined with CTA1-DD we can achieve a safe and above all highly efficacious mucosal adjuvant vector. We optimized the construction, tested the requirements for function and evaluated proof-of-concept in an influenza A virus challenge model. We demonstrated that the CTA1-3M2e-DD/ISCOMS vector provided 100% protection against mortality and greatly reduced morbidity in the mouse model. The immunogenicity of the vector was superior to other vaccine formulations using the ISCOM or CTA1-DD adjuvants alone. The versatility of the vector was best exemplified by the many options to insert, incorporate or admix vaccine antigens with the vector. Furthermore, the CTA1-3M2e-DD/ISCOMS could be kept 1 year at 4°C or as a freeze-dried powder without affecting immunogenicity or adjuvanticity of the vector. Strong serum IgG and mucosal IgA responses were elicited and CD4 T cell responses were greatly enhanced after intranasal administration of the combined vector. Together these findings hold promise for the combined vector as a mucosal vaccine against influenza virus infections including pandemic influenza. The CTA1-DD/ISCOMS technology represents a breakthrough in mucosal vaccine vector design which successfully combines immunomodulation and targeting in a safe and stable particulate formation.

Nasal immunization with plasmid DNA encoding P6 protein and immunostimulatory complexes elicits nontypeable Haemophilus influenzae-specific long-term mucosal immune responses in the nasopharynx.

Nasal vaccination is an effective therapeutic regimen for preventing upper respiratory infection, while DNA vaccines represent a new approach for controlling infectious diseases. Here, we examined the efficacy of nasally administered DNA vaccine on upper respiratory infections. A DNA plasmid encoding the P6 outer membrane protein of nontypeable Haemophilus influenzae (NTHi) was constructed. Mice were immunized 3 times intranasally with the DNA plasmid and Matrix-M, an immunostimulatory complex adjuvant. P6-specific immune responses were examined using purified P6 protein. Nasal-associated lymphoid tissue (NALT) CD4(+) T cells were purified and incubated with feeder cells in the presence of P6, and the expression of cytokine mRNA was examined. In addition, NTHi challenges were performed and the level of NTHi was quantified in nasal washes. P6-specific nasal wash IgA and serum IgG were elevated following immunization with the DNA plasmid and Matrix-M. The number of specific IgA-producing cells increased in the nasal passages of the immunized mice. In addition to Th1 and Th2 cytokine expression, IL-17 was detected in P6-specific NALT CD4(+) T cells. Moreover, DNA vaccination enhanced bacterial clearance. These findings suggest that a successful DNA vaccination protocol has been developed for inducing in vivo immune responses against NTHi. Nasal vaccination with P6 DNA vaccine and Matrix-M might be a new effective regimen for the induction of specific protective immunity in the upper respiratory tract.

A multi-subunit chlamydial vaccine induces antibody and cell-mediated immunity in immunized koalas (Phascolarctos cinereus): comparison of three different adjuvants.

PROBLEM: Chlamydial infections represent a major threat to the survival of the koala. Infections caused by Chlamydia pecorum cause blindness, infertility, pneumonia and urinary tract infections and represent a threat to the survival of the species. Little is known about the immune response in koalas, or the safety of commonly used adjuvants for induction of protective systemic and mucosal immunity. METHOD: of study In the present study, we immunized 18 healthy female koalas subcutaneously with a combination of three chlamydial antigens [major outer membrane protein (MOMP), NrdB and TC0512 (Omp85)] mixed with one of three different adjuvants [Alhydrogel, Immunostimulating Complex (ISC) and TiterMax Gold]. RESULTS: All adjuvants induced strong neutralizing IgG responses in plasma against the three antigens with prolonged responses lasting more than 270 days seen in Alhydrogel and ISC immunized animals. Cloacal IgG responses lasting >270 days were also induced in ISC-immunized animals. Chlamydia-specific peripheral blood mononuclear cell proliferative responses were elicited by both Alhydrogel and ISC, and these lasted >270 days in the ISC group. CONCLUSION: The data show that a multi-subunit chlamydial vaccine, given subcutaneously, can elicit Chlamydia-specific cell-mediated and antibody responses in the koala demonstrating that the development of a protective vaccine is feasible.

Immunostimulatory complexes containing Eimeria tenella antigens and low toxicity plant saponins induce antibody response and provide protection from challenge in broiler chickens.

Immunostimulating complexes (ISCOMs) are unique multimolecular structures formed by encapsulating antigens, lipids and triterpene saponins and are one of the most successful antigen delivery systems for microbial antigens. In the current study, both the route of administration and the antigen concentration of ISCOMs, containing Eimeria tenella antigens and saponins from native plants, were evaluated in their ability to stimulate humoral immunity and to protect chickens against a challenge infection with E. tenella. Broiler chickens were immunized with ISCOM preparations containing E. tenella antigens and the purified saponins Gg6, Ah6 and Gp7 isolated from Glycyrrhiza glabra, Aesculus hippocastanum and Gipsophila paniculata, respectively. The effects of the route of administration, dose of antigen and type of saponin used for construction of ISCOMs were evaluated for ability to stimulate serum IgG and IgM and to protect chickens against a homologous challenge. A single intranasal immunization was the most effective route for administering ISCOMs although the in ovo route was also quite effective. Dose titration experiments demonstrated efficacy after single immunization with various ISCOM doses but maximum effects were observed when ISCOMs contain 5-10mug antigen. Immunization of birds by any of the three routes with E. tenella antigens alone or antigens mixed with alum hydroxide adjuvant resulted in lower serum antibody and reduced protection to challenge relative to immunization with ISCOMs. Overall the results of this study confirm that significant immunostimulation and protection to challenge are achieved by immunization of chickens with ISCOMs containing purified saponins and native E. tenella antigens and suggest that ISCOMs may be successfully used to develop a safe and effective vaccine for prevention of avian coccidiosis.

Adjuvants for pandemic influenza vaccines.

The use of adjuvants is being explored as a means of improving vaccine immunogenicity. This is particularly important for the development of vaccines against potential pandemic influenza virus strains. Adjuvants act by prolonging the exposure time of antigen to the immune system, enhancing the delivery of antigen to antigen-presenting cells, or providing immunostimulatory signals that potentiate the immune response. Aluminum salts are the only licensed adjuvant in the United States, but the combination of these salts with inactivated influenza A/H5N1 antigens has had little effect on seroresponses. Several oil-in-water adjuvants, including MF59 and AS03, have significantly enhanced immune responses in healthy adult vaccine recipients to inactivated influenza A/H5N1. Additional studies are needed in vulnerable populations (younger and elderly persons, pregnant women, and immunocompromised patients) to confirm the safety and enhanced immunogenicity of these promising formulations. A number of other adjuvants are under investigation to evaluate their ability to improve the immunogenicity of inactivated vaccines targeting influenza A/H5N1.

Eimeria tenella: utilization of a nasal vaccine with sporozoite antigens incorporated into Iscom as protection for broiler breeders against a homologous challenge.

The aim of this study was to evaluate a nasal vaccine using antigens derived from sporozoites of Eimeria tenella incorporated into Iscom to protect broiler chicks. Forty-five one-day-old chickens (Cobb), unvaccinated against coccidiosis, were used in this experiment. The birds were maintained in separated battery cages and divided into three groups: G1 (n=15), G2 (n=15), and G3 (n=15). G1 received 50 microg of sporozoites+Iscom vaccine, G2 received Iscom without antigens, and G3 received only PBS. The treatments were administered by nasal route on days 0, 7, and 21 of the experiment. On the 28th day, all birds were challenged with 105 sporulated oocysts of E. tenella. On the challenge day, three birds from each group were euthanized to evaluate lymphocyte proliferation. Lesion scores were obtained from five birds from each group, 7 days after challenge. The remaining animals were euthanized on the 50th day. The mean lymphocyte proliferation responses were significantly different (P=0.03); G1 was 2.3-2.6 times more elevated than G2, and G3 (P<0.001). 83% of the birds from G1 showed an IgY antibody reaction by ELISA at challenge. The means for oocysts shedding were 16,890+/-20,511, 48,080+/-50,047, and 65,020+/-74,461, for G1, G2 and G3 birds, respectively. There was no significant difference (P>0.17) in oocysts shedding between groups. However, the G1 and G2 chicks demonstrated reduction in percentage of oocyst shedding when compared to control birds (G3) by 74.02% and 26.05%, respectively. The average lesion scores were G1=0.4, G2=1, and G3=2. This study demonstrated that the lowest lesion score and oocyst shedding were observed in the birds from the group that received antigens derived from sporozoite with an Iscom adjuvant (G1). These results suggest that this vaccine can induce protection against avian coccidiosis.

Evaluation of ISCOMATRIX and ISCOM vaccines for immunisation against Helicobacter pylori.

An important obstacle to development of an effective vaccine against Helicobacter pylori is the lack of a suitable adjuvant. This study evaluated the effectiveness of ISCOMATRIX and ISCOM vaccines at inducing protective immunity against H. pylori in mice. Immunisation with ISCOMATRIX and ISCOM vaccines resulted in a reduction in H. pylori colonisation equivalent to that induced by the gold standard cholera toxin (CT) adjuvant. Protection was induced in two mouse backgrounds, using two different bacterial antigens and following vaccine delivery via either the intranasal or subcutaneous route. This supports the potential of ISCOMATRIX and ISCOM technologies in H. pylori vaccine development.