Yeast Shells Encapsulating Adjuvant AS04 as an Antigen Delivery System for a Novel Vaccine against Toxoplasma Gondii.

Toxoplasma gondii (T. gondii) infection causes severe zoonotic toxoplasmosis, which threatens the safety of almost one-third of the human population globally. However, there is no effective protective vaccine against human toxoplasmosis. This necessitates anti-T. gondii vaccine development, which is a main priority of public health. In this study, we optimized the adjuvant system 04 (AS04), a vaccine adjuvant constituted by 3-O-desacyl-4'-monophosphoryl lipid A (a TLR4 agonist) and aluminum salts, by packing it within natural extracts of ß-glucan particles (GPs) from Saccharomyces cerevisiae to form a GP-AS04 hybrid adjuvant system. Through a simple mixing procedure, we loaded GP-AS04 particles with the total extract (TE) of T. gondii lysate, forming a novel anti-T. gondii vaccine GP-AS04-TE. Results indicated that the hybrid adjuvant can efficiently and stably load antigens, mediate antigen delivery, facilitate the dendritic uptake of antigens, boost dendritic cell maturation and stimulation, and increase the secretion of pro-inflammatory cytokines. In the mouse inoculation model, GP-AS04-TE significantly stimulated the function of dendritic cells, induced a very strong TE-specific humoral and cellular immune response, and finally showed a strong and effective protection against toxoplasma chronic and acute infections. This work proves the potential of GP-AS04 for exploitation as a vaccine against a range of pathogens.

CpG-adjuvanted stable prefusion SARS-CoV-2 spike protein protected hamsters from SARS-CoV-2 challenge.

The COVID-19 pandemic presents an unprecedented challenge to global public health. Rapid development and deployment of safe and effective vaccines are imperative to control the pandemic. In the current study, we applied our adjuvanted stable prefusion SARS-CoV-2 spike (S-2P)-based vaccine, MVC-COV1901, to hamster models to demonstrate immunogenicity and protection from virus challenge. Golden Syrian hamsters immunized intramuscularly with two injections of 1 µg or 5 µg of S-2P adjuvanted with CpG 1018 and aluminum hydroxide (alum) were challenged intranasally with SARS-CoV-2. Prior to virus challenge, the vaccine induced high levels of neutralizing antibodies with 10,000-fold higher IgG level and an average of 50-fold higher pseudovirus neutralizing titers in either dose groups than vehicle or adjuvant control groups. Six days after infection, vaccinated hamsters did not display any weight loss associated with infection and had significantly reduced lung pathology and most importantly, lung viral load levels were reduced to lower than detection limit compared to unvaccinated animals. Vaccination with either 1 µg or 5 µg of adjuvanted S-2P produced comparable immunogenicity and protection from infection. This study builds upon our previous results to support the clinical development of MVC-COV1901 as a safe, highly immunogenic, and protective COVID-19 vaccine.

Safety and immunogenicity of a synthetic carbohydrate conjugate vaccine against Shigella flexneri 2a in healthy adult volunteers: a phase 1, dose-escalating, single-blind, randomised, placebo-controlled study.

BACKGROUND: Shigella remains in the top four pathogens responsible for moderate to severe diarrhoea in children below 5 years of age. The shigella O-specific polysaccharide (O-SP) is a promising vaccine target. We developed a conjugate vaccine prototype incorporating a unique well defined synthetic oligosaccharide hapten, chemically designed for optimal antigenic, conformational, structural, and functional mimicry of the O-SP from Shigella flexneri 2a (SF2a). We aimed to assess the safety, tolerability, and immunogenicity of this original synthetic oligosaccharide-based vaccine candidate, SF2a-TT15, conceived to drive the antibody response towards the key protective determinants of the native lipopolysaccharide antigen, in a first-in-human phase 1 study. METHODS: We did a first-in-human, dose-escalating, single-blind, observer-masked, randomised, placebo-controlled study at the Clinical Research Center of Tel Aviv Sourasky Medical Center (Israel). Participants were healthy adults aged 18-45 years with low titres of serum SF2a-specific IgG antibodies. 64 eligible participants were assigned to one of two cohorts. 32 participants in each of the two cohorts were randomly assigned via computer-generated algorithm in a stepwise manner to receive the 2 µg (cohort 1) and 10 µg oligosaccharide dose (cohort 2) of the SF2a-TT15 vaccine candidate non-adjuvanted or adjuvanted with aluminium hydroxide (alum) or matching placebos. The vaccine was administered as three single intramuscular injections into the arm, 28 days apart. The primary outcome was the incidence and severity of adverse events, which were assessed in the intention-to-treat safety population analysis including all participants who were randomly assigned and received at least one vaccine or placebo injection. The immunogenicity endpoints were secondary outcomes and were analysed in all participants who were randomly assigned, received all of the assigned injections before the time of the immunogenicity assessment, and provided blood samples for immunological follow-up (per-protocol immunogenicity analysis). The study is registered with ClinicalStudies.gov, NCT02797236 and is completed. FINDINGS: Of 203 volunteers initially screened, 64 participants were enrolled between Sept 20, 2016, and Sept 26, 2017. In each of the two cohorts, 12 participants received the adjuvanted vaccine, 12 received the non-adjuvanted vaccine and eight received the matching placebo (four each). The SF2a-TT15 glycoconjugate was well tolerated at both doses. No serious or severe adverse events occurred. Overall, seven (88%) of eight to 12 (100%) of 12 in each group of volunteers had one adverse event or more after receiving the study agents with the majority of adverse events, 300 (98%) of 307, considered mild in intensity. Of the seven adverse events defined as moderate in severity, one (nausea) was suspected to be related to the vaccine candidate. At all post-immunisation days and for both oligosaccharide doses, whether adjuvanted or not, SF2a-TT15 induced significantly higher serum IgG anti-SF2a lipopolysaccharide geometric mean titres (GMTs) as compared with baseline or with the corresponding GMTs in placebo recipients (p<0·01). After one injection, the non-adjuvanted 10 µg oligosaccharide dose induced a 27-times increase in IgG GMT (5080 vs 189) and the non-adjuvanted 2 µg oligosaccharide dose induced a five-times increase (1411 vs 283), compared with baseline. Alum enhanced the specific IgG response at 2 µg oligosaccharide dose after the third injection (GMTs 3200 vs 1176, p=0.045). INTERPRETATION: SF2a-TT15 was safe and well tolerated and induced high titres of anti-SF2a LPS IgG antibodies. These results support further evaluation of this original synthetic oligosaccharide-protein conjugate vaccine candidate for safety, immunogenicity, and protective efficacy in target populations. FUNDING: The European Union Seventh Framework Programme.

A natural oil increases specific anti-OVA IgG levels and induces a cellular immune response combined with aluminum hydroxide.

The efficacy of certain vaccines is improved by the use of adjuvants. Nowadays, the development of new, effective, and safe adjuvants that stimulate the innate immune response is researched. In this context, medicinal plants appear as a suitable alternative. Minthostachys verticillata essential oil (EO) has demonstrated the ability to modulate mechanisms of the innate immune response. Thus, the present work aimed to evaluate the EO adjuvant effect on humoral and cellular immunity, coadministered with OVA as antigen. The chemical analysis of EO by gas chromatography-mass spectrometry revealed a predominant pulegone-menthone chemotype. EO (1.25, 2.5, or 5.0 mg/ml) did not alter the viability of murine fibroblasts (3T3 cell line) neither showed signs of toxicity in Balb/c mice inoculated subcutaneously. The serum of mice immunized with OVA + EO showed increased levels of anti-OVA-specific antibodies of IgG1 subclass compared with the mice immunized with OVA alone revealing an adjuvant effect of EO. The delayed type hypersensitivity showed that the combination OVA + Al(OH)3  + EO was the best to induce a cellular immune response that extended until 48 h postinjection of OVA. M. verticillata EO appears as a new, safe, and effective adjuvant, which should continue to be studied for their possible future incorporation into vaccine formulations.

CoVaccine HT™ Adjuvant Potentiates Robust Immune Responses to Recombinant SARS-CoV-2 Spike S1 Immunization.

The current COVID-19 pandemic has claimed hundreds of thousands of lives and its causative agent, SARS-CoV-2, has infected millions, globally. The highly contagious nature of this respiratory virus has spurred massive global efforts to develop vaccines at record speeds. In addition to enhanced immunogen delivery, adjuvants may greatly impact protective efficacy of a SARS-CoV-2 vaccine. To investigate adjuvant suitability, we formulated protein subunit vaccines consisting of the recombinant S1 domain of SARS-CoV-2 Spike protein alone or in combination with either CoVaccine HT™ or Alhydrogel. CoVaccine HT™ induced high titres of antigen-binding IgG after a single dose, facilitated affinity maturation and class switching to a greater extent than Alhydrogel and elicited potent cell-mediated immunity as well as virus neutralizing antibody titres. Data presented here suggests that adjuvantation with CoVaccine HT™ can rapidly induce a comprehensive and protective immune response to SARS-CoV-2.

Immunogenicity of Antigen Adjuvanted with AS04 and Its Deposition in the Upper Respiratory Tract after Intranasal Administration.

Adjuvant system 04 (AS04) is in injectable human vaccines. AS04 contains two known adjuvants, 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and insoluble aluminum salts. Data from previous studies showed that both MPL and insoluble aluminum salts have nasal mucosal vaccine adjuvant activity. The present study was designed to test the feasibility of using AS04 as an adjuvant to help nasally administered antigens to induce specific mucosal and systemic immunity as well as to evaluate the deposition of antigens in the upper respiratory tract when adjuvanted with AS04. Alhydrogel, an aluminum (oxy)hydroxide suspension, was mixed with MPL to form AS04, which was then mixed with ovalbumin (OVA) or 3× M2e-HA2, a synthetic influenza virus hemagglutinin fusion protein, as an antigen to prepare OVA/AS04 and 3× M2e-HA2/AS04 vaccines, respectively. In mice, AS04 enabled antigens, when given intranasally, to induce specific IgA response in nasal and lung mucosal secretions as well as specific IgG response in the serum samples of the immunized mice, whereas subcutaneous injection of the same vaccine induced specific antibody responses only in the serum samples but not in the mucosal secretions. Splenocytes isolated from mice intranasally immunized with the OVA/AS04 also proliferated and released cytokines (i.e., IL-4 and IFN-γ) after in vitro stimulation with the antigen. In the immunogenicity test, intranasal OVA/AS04 was not more effective than intranasal OVA/MPL at the dosing regimens tested. However, when compared to OVA/MPL, OVA/AS04 showed a different atomized droplet size distribution and more importantly a more favorable OVA deposition profile when atomized into a nasal cast that was 3-D printed based on the computer tomography scan of the nose of a child. It is concluded that AS04 has mucosal adjuvant activity when given intranasally. In addition, there is a reason to be optimistic about using AS04 as an adjuvant to target an antigen of interest to the right region of the nasal cavity in humans for immune response induction.

Crystalline and Amorphous Preparation of Aluminum Hydroxide Nanoparticles Enhances Protective Antigen Domain 4 Specific Immunogenicity and Provides Protection Against Anthrax.

INTRODUCTION: Aluminum salts, although they have been used as adjuvants in many vaccine formulations since 1926, exclusively induce a Th2-biased immune response, thereby limiting their use against intracellular pathogens like Mycobacterium tuberculosis. METHODS AND RESULTS: Herein, we synthesized amorphous and crystalline forms of aluminum hydroxide nanoparticles (AH nps) of 150-200 nm size range. Using Bacillus anthracis protective antigen domain 4 (D4) as a model antigen, we demonstrated that both amorphous and crystalline forms of AH nps displayed enhanced antigen D4 uptake by THP1 cells as compared to commercial adjuvant aluminum hydroxide gel (AH gel). In a mouse model, both amorphous and crystalline AH nps triggered an enhanced D4-specific Th2- and Th1-type immune response and conferred superior protection against anthrax spore challenge as compared to AH gel. Physicochemical characterization of crystalline and amorphous AH nps revealed stronger antigen D4 binding and release than AH gel. CONCLUSION: These results demonstrate that size and crystallinity of AH nps play important roles in mediating enhanced antigen presenting cells (APCs) activation and potentiating a strong antigen-specific immune response, and are critical parameters for the rational design of alum-based Th1-type adjuvant to induce a more balanced antigen-specific immune response.

PRIMVAC vaccine adjuvanted with Alhydrogel or GLA-SE to prevent placental malaria: a first-in-human, randomised, double-blind, placebo-controlled study.

BACKGROUND: PRIMVAC is a VAR2CSA-derived placental malaria vaccine candidate aiming to prevent serious clinical outcomes of Plasmodium falciparum infection during pregnancy. We assessed the safety and immunogenicity of PRIMVAC adjuvanted with Alhydrogel or glucopyranosyl lipid adjuvant in stable emulsion (GLA-SE) in French and Burkinabe women who were not pregnant. METHODS: This first-in-human, randomised, double-blind, placebo-controlled, dose escalation trial was done in two staggered phases, a phase 1A trial in 18-35-year-old women who were malaria naive in a hospital in France and a subsequent phase 1B trial in women who were naturally exposed to P falciparum and nulligravid in the clinical site of a research centre in Burkina Faso. Volunteers were recruited into four sequential cohorts receiving PRIMVAC intramuscularly at day 0, 28, and 56: two cohorts in France receiving 20 µg or 50 µg of PRIMVAC and then two in Burkina Faso receiving 50 µg or 100 µg of PRIMVAC. Volunteers were randomly assigned (1:1) to two groups (PRIMVAC adjuvanted with either Alhydrogel or GLA-SE) in France and randomly assigned (2:2:1) to three groups (PRIMVAC adjuvanted with either Alhydrogel, GLA-SE, or placebo) in Burkina Faso. Randomisation was centralised, using stratification by cohort and blocks of variable size, and syringes were masked by opaque labels. The primary endpoint was the proportion of participants with any grade 3 or higher adverse reaction to vaccination up until day 35. Safety at later time points as well as humoral and cellular immunogenicity were assessed in secondary endpoints. This trial is registered with ClinicalTrials.gov, NCT02658253. FINDINGS: Between April 19, 2016, and July 13, 2017, 68 women (18 in France, 50 in Burkina Faso) of 101 assessed for eligibility were included. No serious adverse event related to the vaccine occurred. PRIMVAC antibody titres increased with each dose and seroconversion was observed in all women vaccinated with PRIMVAC (n=57). PRIMVAC antibody titres reached a peak (geometric mean 11 843·0, optical density [OD] 1·0, 95% CI 7559·8-18 552·9 with 100 µg dose and GLA-SE) 1 week after the third vaccination (day 63). Compared with Alhydrogel, GLA-SE tended to improve the PRIMVAC antibody response (geometric mean 2163·5, OD 1·0, 95% CI 1315·7-3557·7 with 100 µg dose and Alhydrogel at day 63). 1 year after the last vaccination, 20 (71%) of 28 women who were vaccinated with PRIMVAC/Alhydrogel and 26 (93%) of 28 women who were vaccinated with PRIMVAC/GLA-SE still had anti-PRIMVAC antibodies, although antibody magnitude was markedly lower (452·4, OD 1·0, 95% CI 321·8-636·1 with 100 µg dose and GLA-SE). These antibodies reacted with native homologous VAR2CSA expressed by NF54-CSA infected erythrocytes (fold change from baseline at day 63 with 100 µg dose and GLA-SE: 10·74, 95% CI 8·36-13·79). Limited cross-recognition, restricted to sera collected from women that received the 100 µg PRIMVAC dose, was observed against heterologous VAR2CSA variants expressed by FCR3-CSA (fold change from baseline at day 63: 1·49, 95% CI 1·19-1·88) and 7G8-CSA infected erythrocytes (1·2, 1·08-1·34). INTERPRETATION: PRIMVAC adjuvanted with Alhydrogel or GLA-SE had an acceptable safety profile, was immunogenic, and induced functional antibodies reacting with the homologous VAR2CSA variant expressed by NF54-CSA infected erythrocytes. Cross-reactivity against heterologous VAR2CSA variants was limited and only observed in the higher dose group. An alternate schedule of immunisation, antigen dose, and combinations with other VAR2CSA-based vaccines are envisaged to improve the cross-reactivity against heterologous VAR2CSA variants. FUNDING: Bundesministerium für Bildung und Forschung, through Kreditanstalt für Wiederaufbau, Germany; Inserm, and Institut National de Transfusion Sanguine, France; Irish Aid, Department of Foreign Affairs and Trade, Ireland.

Immunogenicity and safety of an adjuvanted inactivated polio vaccine, IPV-Al, compared to standard IPV: A phase 3 observer-blinded, randomised, controlled trial in infants vaccinated at 6, 10, 14 weeks and 9 months of age.

BACKGROUND: A dose-sparing inactivated polio vaccine (IPV-Al), obtained by adsorption of inactivated virus to an aluminium hydroxide adjuvant, can help mitigate global supply and the cost constraints of IPV. The objective of this trial was to demonstrate the non-inferiority of IPV-Al to standard IPV. METHODS: This phase 3, observer-blinded, randomised, controlled trial was conducted at 5 investigational sites in the Philippines. Infants not previously vaccinated with any polio vaccines were randomised to receive three IPV-Al (n = 502) or IPV vaccinations (n = 500) at 6, 10 and 14 weeks of age plus a booster vaccination at 9 months. The primary endpoint was type-specific seroconversion, defined as an antibody titre ≥4-fold higher than the estimated maternal antibody titre and a titre ≥8, one month after the primary vaccination series. RESULTS: Seroconversion rates following primary vaccination with IPV-Al (483 infants in the per-protocol analysis set) or IPV (478 infants) were: polio type 1, 97.1% versus 99.0%; type 2, 94.2% versus 99.0%; and type 3, 98.3% versus 99.6%. IPV-Al was non-inferior to IPV, as the lower 95% confidence limits of the treatment differences were above the predefined -10%-point limit: type 1, -1.85% (-3.85; -0.05); type 2, -4.75% (-7.28; -2.52); type 3, -1.24 (-2.84; 0.13). The booster effect (geometric mean titre (GMT) post-booster / GMT pre-booster) was: type 1, 63 versus 43; type 2, 54 versus 47; type 3, 112 versus 80. IPV-Al was well tolerated with a safety profile comparable to that of IPV. Serious adverse events were recorded for 29 infants (5.8%, 37 events) in the IPV-Al group compared to 28 (5.6%, 48 events) in the IPV group. CONCLUSION: Non-inferiority of IPV-Al to IPV with respect to seroconversion was confirmed and a robust booster response was demonstrated. Both vaccines had a similar safety profile. ClinicalTrials.gov identifier: NCT03032419.

Accelerated Dose Escalation with Three Injections of an Aluminum Hydroxide-Adsorbed Allergoid Preparation of Six Grasses Is Safe for Patients with Moderate to Severe Allergic Rhinitis.

Only few data on safety during high-dose, accelerated escalation schedules during subcutaneous allergen immunotherapy (AIT) are available. The aim of this study was to assess the safety and tolerability of an accelerated dose escalation schedule of AIT in adult patients with moderate to severe seasonal rhinoconjunctivitis in a multicenter, open-label, randomized phase II trial. The dose escalation scheme for patients in Group I (1 strength) included 3 injections with 1 strength, B (10,000 TU/mL), whereas the dose escalation scheme for Group II (standard) included 7 injections with 2 strengths, A (1,000 TU/mL) and B (10,000 TU/mL), of an aluminum hydroxide-adsorbed allergoid grass pollen preparation. Overall, 72 of 87 randomized patients (83.7%) reported at least 1 treatment-emergent adverse event (TEAE; 82.2 [Group I] vs. 85.4% [Group II]); 58.8% of all reported TEAEs were assessed as being related to AIT (60.0 vs. 48.8%). The most frequently reported AIT-related TEAEs were swelling (46.7 vs. 34.1%), erythema (28.9 vs. 36.6%), and pruritus (31.1 vs. 17.1%) at the site of the injection. Systemic allergic reactions occurred in 5 (5.8%) patients overall, with more being reported in the 1-strength group (4 [8.9%] vs. 1 [2.4%]). All systemic allergic reactions were classified as World Allergy Organization (WAO) Grade 1 or Grade 2 reactions. Accelerated high-dose escalation with an aluminum hydroxide-adsorbed grass pollen allergoid can be initiated with a safety and tolerability profile comparable to the standard dose escalation schedule in patients with allergic rhinitis with or without asthma.

Effects of lentivirus-mediated CCR3 RNA interference on the function of mast cells of allergic rhinitis in mice.

The CC chemokine receptor 3 (CCR3) expressed by eosinophils, mast cells and Th2 cells is closely related to allergic diseases. The objective of this study was to explore whether silencing of CCR3 with short hairpin RNAs (shRNAs) delivered by a lentiviral vector could impact the function of mast cells in a murine model of allergic rhinitis (AR) in vivo. The murine model of allergic rhinitis (AR) inducing by ovalbumin (OVA) was constructed, and the BALB/c mice were divided into normal control group, AR group, controlshRNA treated group and lentiviral CCR3-shRNA treated group. The recombinant lentivirus vectors which express a short hairpin RNA (shRNA) targeting the CCR3 were dropped into the nasal cavity of OVA-sensitized mice before the challenges. Real-time fluorescence quantitative PCR and western blotting were performed to observe inhibitory effect of CCR3 gene. Nasal symptoms of mice and OVA-specific IgE in each group were assessed. Concentrations of histamine, tryptase and Prostaglandin D2 (PGD2) in bone marrow, peripheral blood and nasal mucosa were analyzed. Furthermore, histological analysis and electron microscopy analysis were applied to detect the histology changes of nasal mucosa and the infiltration of mast cells in nasal mucosa. The results showed that administration of CCR3shRNA could effectively inhibit the expression of the CCR3 gene in bone marrow, peripheral blood and nasal mucosa, which reduced the nasal symptoms, the level of OVA-specific IgE, the inflammatory cells and mast cells infiltration into nasal cavity, and relieved the histopathological changes of nasal mucosa. In addition, intervention of CCR3shRNA could reduce the levels of the histamine, tryptase and PGD2 in bone marrow, peripheral blood and nasal mucosa. These results suggest that inhibition of CCR3 gene expression by shRNAs lentiviral vectors can effectively attenuate migration, infiltration and degranulation of mast cells in local tissues and alleviate the inflammation of allergic rhinitis mice.

Adjuvant effect of TLR7 agonist adsorbed on aluminum hydroxide (AS37): A phase I randomized, dose escalation study of an AS37-adjuvanted meningococcal C conjugated vaccine.

An adjuvant system (AS37) has been developed containing a synthetic toll-like receptor agonist (TLR7a). We conducted a phase I randomized, observer-blind, dose-escalation study to assess the safety and immunogenicity of an investigational AS37-adjuvanted meningococcus C (MenC) conjugate vaccine in healthy adults (NCT02639351). A control group received a licensed MenC conjugate alum-adjuvanted vaccine. Eighty participants were randomized to receive one dose of control or investigational vaccine containing AS37 (TLR7a dose 12.5, 25, 50, 100 µg). All vaccines were well tolerated, apart from in the TLR7a 100 µg dose group, which had three reports (18.8%) of severe systemic adverse events. Four weeks after vaccination, human complement serum bactericidal assay seroresponse rates against MenC were 56-81% in all groups, and ELISA seroresponses were ≥81% for all AS37-adjuvanted vaccine groups (100% in 50 and 100 µg dose groups) and 88% in the control group. Antibody responses were maintained at six months after vaccination.

One-pot synthesis and characterization of ovalbumin-conjugated gold nanoparticles: A comparative study of adjuvanticity against the physical mixture of ovalbumin and gold nanoparticles.

Only few adjuvants are licensed for use in humans and there is a need to develop safe and improved vaccine adjuvants. In this study, we report the one-pot synthesis of antigen ovalbumin (OVA)-conjugated gold nanoparticles (OVA@GNPs). A systematical study was performed by comparing OVA@GNPs with the simple mixture of OVA and gold nanoparticles (OVA+GNPs), including their physiochemical properties through spectrometric and electrophoretic analysis, in vitro stability, cytotoxicity and cellular uptake, and in vivo humoral immune responses following subcutaneous and transcutaneous immunization in mice. The results demonstrate a much stronger interaction between protein and GNPs in OVA@GNPs than OVA+GNPs, which makes OVA@GNPs more stable under in vitro conditions than OVA+GNPs with the ability to induce 4 times higher OVA-specific serum IgG titers following subcutaneous immunization. We also show the dose sparing of OVA@GNPs, as the dosage for aluminum adjuvant required to reach to an equivalent OVA-specific antibody titer was almost five times higher than OVA@GNPs. However, we found that the co-administration of small-sized GNPs had a limited ability for the transcutaneous delivery of OVA. These results demonstrate the potential application of one-pot synthesis approach for producing antigen protein-conjugated gold nanoparticles for vaccine delivery.

Chemical and Immunological Characteristics of Aluminum-Based, Oil-Water Emulsion, and Bacterial-Origin Adjuvants.

Adjuvants are a diverse family of substances whose main objective is to increase the strength, quality, and duration of the immune response caused by vaccines. The most commonly used adjuvants are aluminum-based, oil-water emulsion, and bacterial-origin adjuvants. In this paper, we will discuss how the election of adjuvants is important for the adjuvant-mediated induction of immunity for different types of vaccines. Aluminum-based adjuvants are the most commonly used, the safest, and have the best efficacy, due to the triggering of a strong humoral response, albeit generating a weak induction of cell-mediated immune response. Freund's adjuvant is the most widely used oil-water emulsion adjuvant in animal trials; it stimulates inflammation and causes aggregation and precipitation of soluble protein antigens that facilitate the uptake by antigen-presenting cells (APCs). Adjuvants of bacterial origin, such as flagellin, E. coli membranes, and monophosphoryl lipid A (MLA), are known to potentiate immune responses, but their safety and risks are the main concern of their clinical use. This minireview summarizes the mechanisms that classic and novel adjuvants produce to stimulate immune responses.

Development of FAcE (Formulated Alhydrogel competitive ELISA) method for direct quantification of OAg present in Shigella sonnei GMMA-based vaccine and its optimization using Design of Experiments approach.

Many formulated vaccines, including 1790GAHB Shigella sonnei GMMA-based vaccine, contain Alhydrogel (aluminum hydroxide), consequently the antigen content must be determined in the formulated final vaccine product, as required by regulatory authorities. The direct quantification of antigens adsorbed on aluminum salts is difficult, and antigens may need to be extracted using laborious and often ineffective desorption procedures. To directly quantify the sugar vaccine target in the LPS of 1790GAHB, we have developed a new FAcE (Formulated Alhydrogel competitive ELISA) method. FAcE is an immunoassay based on the competition between S. sonnei LPS, coated on the ELISA plate, and the LPS in formulated S. sonnei GMMA, in binding a specific monoclonal antibody. To optimize the method, which is as easy to perform as a standard ELISA, we have applied a Design of Experiments (DOE) approach. A model was found to define the significant assay variables and to predict their impact on the output responses. Results obtained using the DOE optimized FAcE assay showed that the method is sensitive (0.02 µg/mL lower detection limit), precise, reproducible and can accurately quantify independently formulated drug products, making it a useful tool in routine tests of Alhydrogel-based vaccines. We are currently using this method to determine S. sonnei vaccine potency, stability and lot-to-lot variations, and are broadening its applicability to quantify active ingredients of other Alhydrogel GMMA-vaccines and in multivalent vaccines formulations.

Formulation of aluminum hydroxide adjuvant with TLR agonists poly(I:C) and CpG enhances the magnitude and avidity of the humoral immune response.

Subunit vaccines generally require adjuvants to achieve optimal immune responses. Toll-like receptor (TLR) agonists are promising immune potentiators, but rapid diffusion from the injection site reduces their local effective concentration and may cause systemic reactions. In this study, we investigated the potential of aluminum hydroxide adjuvant (AH) to adsorb the TLR3 agonist poly(I:C) and TLR9 agonist CpG and compared the effect of the combination adjuvant on the immune response with either the TLR agonists or AH alone in mice. Poly(I:C) and CpG readily adsorbed onto AH and this combination adjuvant induced a stronger IgG1 and IgG2a immune response with a significant increase of antibody avidity. The combination adjuvant enhanced antigen uptake and activation of dendritic cells in vitro. It induced an inflammatory response at the injection site similar to AH but without eosinophils which are typically observed with AH. A distinctive antigen-containing monocyte/macrophage population with an intermediate level of CD11c expression was identified in the draining lymph nodes after immunization with TLR agonists and the combination adjuvant. Injection of the combination adjuvant did not induce an increase of TNFα and CXCL10 in serum in contrast to the injection of soluble TLR agonists. These results indicate that this combination adjuvant is a promising formulation to solve some of the unmet needs of current vaccines.

Thermostable Ebola virus vaccine formulations lyophilized in the presence of aluminum hydroxide.

No United States Food and Drug Administration-licensed vaccines protective against Ebola virus (EBOV) infections are currently available. EBOV vaccine candidates currently in development, as well as most currently licensed vaccines in general, require transport and storage under a continuous cold chain in order to prevent potential decreases in product efficacy. Cold chain requirements are particularly difficult to maintain in developing countries. To improve thermostability and reduce costly cold chain requirements, a subunit protein vaccine against EBOV was formulated as a glassy solid using lyophilization. Formulations of the key antigen, Ebola glycoprotein (EBOV-GP), adjuvanted with microparticulate aluminum hydroxide were prepared in liquid and lyophilized forms, and the vaccines were incubated at 40 °C for 12 weeks. Aggregation and degradation of EBOV-GP were observed in liquid formulations during the 12-week incubation period, whereas changes were minimal in lyophilized formulations. Antibody responses against EBOV-GP following three intramuscular immunizations in BALB/c mice were used to determine vaccine immunogenicity. EBOV-GP formulations were equally immunogenic in liquid and lyophilized forms. After lyophilization and reconstitution, adjuvanted vaccine formulations produced anti-EBOV-GP IgG antibody responses in mice similar to those generated against corresponding adjuvanted liquid vaccine formulations. More importantly, antibody responses in mice injected with reconstituted lyophilized vaccine formulations that had been incubated at 40 °C for 12 weeks prior to injection indicated that vaccine immunogenicity was fully retained after high-temperature storage, showing promise for future vaccine development efforts.

A comparison between adjuvant and delivering functions of calcium phosphate, aluminum hydroxide and chitosan nanoparticles, using a model protein of Brucella melitensis Omp31.

Vaccination is the most efficient and economic approach used to hinder infection and intense consequences caused by viruses, bacteria, or other pathogenic organisms. Since the intrinsic immunogenicity of recombinant antigens is usually low, safe and potent vaccine adjuvants are needed to ensure the success of those recombinant vaccines. Nanoparticles (NPs) have attracted much interest as adjuvants and delivery systems. Previous studies have shown that calcium phosphate (CP), aluminum hydroxide (AH) and chitosan (CS) NPs are promising delivery systems for immunization. In addition, it has been determined that Omp31 is a good candidate for inducing protection against Brucella (B) melitensis and B. ovis. Our aim in the present study was to compare the functions of CP, AH and CS NPs for stimulation of the immune response and protection against B. melitensis by using omp31 as a model protein. Based on the cytokine profile and subclasses of the antibody, vaccination with Omp31 load CP (CP/Omp31) and Omp31 load AH (AH/Omp31) NPs induced T helper type 1 (Th1)-T helper type 2 (Th2) immune response, whereas immunization by Omp31 load CS (CS/Omp31) NPs induced Th1 immune response. CP/Omp31 NPs elicited protection toward B. melitensis challenge equivalent to the vaccine strain B. melitensis Rev.1. Compared to CS/Omp31 NPs, CP/Omp31 NPs elicited a low increase in protection level against B. melitensis 16 M. In conclusion, the obtained results indicated that CP NPs were potent antigen delivery systems to immunize brucellosis.

Immunogenicity of HPV and HBV vaccines: adjuvanticity of synthetic analogs of monophosphoryl lipid A combined with aluminum hydroxide.

Monophosphoryl lipid A (MPL), a purified and detoxified product of lipopolysaccharide (LPS) of Salmonella minnesota R595, has been used as an adjuvant in different vaccines. In this study, the efficacy of human papillomaviruses (HPV) and hepatitis B virus (HBV) vaccines formulated with aluminum hydroxide combined with two different synthetic MPLs, 3D-(6-acyl)-PHAD or 3D-PHAD, or aluminum hydroxide combined with the mixtures of such MPLs, has been assessed. The immunogenicity in female BALB/c mice was verified by two intramuscular injections of differently formulated HPV and HBV vaccines and the total immunoglobulin G (IgG) antibody response was considered to compare the employed adjuvants. As verified experimentally, a mixture of 3D-(6-acyl)-PHAD and 3D-PHAD was able to induce significantly higher antibody titer than that of either 3D-(6-acyl)-PHAD or 3D-PHAD, when used individually. Interestingly, based on the responses achieved in terms of the total antibody levels, such mixture of synthetic MPLs was found to be even more effective than the bacterially derived MPL. Accordingly, the obtained results indicated that, if designed appropriately, synthetic MPL molecules could provide improved adjuvanticity with high level of consistency.

Chronic helminth infection does not impair immune response to malaria transmission blocking vaccine Pfs230D1-EPA/Alhydrogel® in mice.

INTRODUCTION: Malaria transmission blocking vaccines (TBV) are innovative approaches that aim to induce immunity in humans against Plasmodium during mosquito stage, neutralizing the capacity of the infected vectors to transmit malaria. Pfs230D1-EPA/Alhydrogel®, a promising protein-protein conjugate malaria TBV, is currently being tested in human clinical trials in areas where P. falciparum malaria is coendemic with helminth parasites. Helminths are complex metazoans that share the master capacity to downregulate the host immune response towards themselves and also to bystander antigens, including vaccines. However, it is not known whether the activity of a protein-based malaria TBV may be affected by a chronic helminth infection. METHODS: Using an experimental murine model for a chronic helminth infection (Heligmosomoides polygyrus bakeri - Hpb), we evaluated whether prior infection alters the activity of Pfs230D1-EPA/Alhydrogel® TBV in mice. RESULTS: After establishment of a chronic infection, characterized by a marked increase of parasite antigen-specific IgG1, IgA and IgE antibody responses, concomitant with an increase of systemic IL-10, IL-5 and IL-6 levels, the Hpb-infected mice were immunized with Pfs230D1-EPA/Alhydrogel® and the vaccine-specific immune response was compared with that in non-infected immunized mice. TBV immunizations induced an elevated vaccine specific-antibody response, however Pfs230D1 specific-IgG levels were similar between infected and uninfected mice at days 15, 25 and 35 post-vaccination. Absolute numbers of Pfs230D1-activated B cells generated in response to the vaccine were also similar among the vaccinated groups. Finally, vaccine activity assessed by reduction of oocyst number in P. falciparum infected mosquitoes was similar between Hpb-infected and immunized mice with non-infected immunized mice. CONCLUSION: Pfs230D1-EPA/Alhydrogel® efficacy is not impaired by a chronic helminth infection in mice.