AddaVax Adjuvant Research Articles

CpG 1018 Is an Effective Adjuvant for Influenza Nucleoprotein.

Current influenza vaccines mainly induce neutralizing antibodies against the highly variable surface antigen hemagglutinin and require annual manufacturing and immunization. Different from surface antigens, intracellular nucleoprotein (NP) is highly conserved and has been an attractive target to develop universal T cell vaccines against influenza. Yet, influenza NP protein mainly induces humoral immune responses and lacks the ability to induce potent cytotoxic T lymphocyte (CTL) responses, key for the success of universal T cell vaccines. This study compared CpG 1018 and AddaVax to enhance recombinant NP-induced CTL responses and protection in murine models. CpG 1018 was explored to boost intradermal NP immunization, while AddaVax was explored to boost intramuscular NP immunization due to the high risk of AddaVax adjuvant to induce significant local reactions following intradermal delivery. We found CpG 1018 was highly effective to enhance NP-induced humoral and cellular immune responses superior to AddaVax adjuvant. Furthermore, CpG 1018 potentiated Th1-biased antibody responses, while AddaVax enhanced Th1/Th2-balanced antibody responses. CpG 1018 significantly enhanced IFNγ-secreting Th1 cells, while AddaVax adjuvant significantly increased IL4-secreting Th2 cells. Influenza NP immunization in the presence of CpG 1018 induced significant protection against lethal viral challenges, while influenza NP immunization in the presence of AddaVax failed to elicit significant protection. Our data validated CpG 1018 as an effective adjuvant to enhance influenza NP-induced CTL responses and protection.

S Trimer Derived from SARS-CoV-2 B.1.351 and B.1.618 Induced Effective Immune Response against Multiple SARS-CoV-2 Variants

The spread of SARS-CoV-2 and its variants leads to a heavy burden on healthcare and the global economy, highlighting the need for developing vaccines that induce broad immunity against coronavirus. Here, we explored the immunogenicity of monovalent or bivalent spike (S) trimer subunit vaccines derived from SARS-CoV-2 B.1.351 (S1-2P) or/and B.1. 618 (S2-2P) in Balb/c mice. Both S1-2P and S2-2P elicited anti-spike antibody responses, and alum adjuvant induced higher levels of antibodies than Addavax adjuvant. The dose responses of the vaccines on immunogenicity were evaluated in vivo. A low dose of 5 μg monovalent recombinant protein or 2.5 μg bivalent vaccine triggered high-titer antibodies that showed cross-activity to Beta, Delta, and Gamma RBD in mice. The third immunization dose could boost (1.1 to 40.6 times) high levels of cross-binding antibodies and elicit high titers of neutralizing antibodies (64 to 1024) prototype, Beta, Delta, and Omicron variants. Furthermore, the vaccines were able to provoke a Th1-biased cellular immune response. Significantly, at the same antigen dose, S1-2P immune sera induced stronger broadly neutralizing antibodies against prototype, Beta, Delta, and Omicron variants compared to that induced by S2-2P. At the same time, the low dose of bivalent vaccine containing S2-2P and S1-2P (2.5 μg for each antigen) significantly improved the cross-neutralizing antibody responses. In conclusion, our results showed that monovalent S1-2P subunit vaccine or bivalent vaccine (S1-2P and S2-2P) induced potent humoral and cellular responses against multiple SARS-CoV-2 variants and provided valuable information for the development of recombinant protein-based SARS-CoV-2 vaccines that protect against emerging SARS-CoV-2 variants.

Functional characterization and immunogenicity of a novel vaccine candidate against tick-borne encephalitis virus based on Leishmania-derived virus-like particles

Tick-borne encephalitis virus (TBEV) is a major cause of neurological infections in many regions of central, eastern and northern Europe and northern Asia. In approximately 15% of cases, TBEV infections lead to the development of severe encephalitis or meningitis. The main route of TBEV transmission is tick bites; however, ingestion of dairy products from infected animals (goats, cattle and sheep) is also a frequent cause of the disease. Therefore, vaccination of livestock in virus endemic regions could also contribute to the decrease in TBEV infection among humans. Although few vaccines against TBEV based on inactivated viruses are available for humans, due to high costs, vaccination is not mandatory in most of the affected countries. Moreover, there is still no vaccine for veterinary use. Here, we present a characterization and immunogenicity study of a new potential TBEV vaccine based on virus-like particles (VLPs) produced in Leishmania tarentolae cells. VLPs, which mimic native viral particles but do not contain genetic material, show good immunogenic potential. For the first time, we showed that the protozoan L. tarentolae expression system can be successfully used for the production of TBEV virus-like particles with highly efficient production. We confirmed that TBEV recombinant structural proteins (prM/M and E) from VLPs are highly recognized by neutralizing antibodies in in vitro analyses. Therefore, VLPs in combination with AddaVax adjuvant were used in immunization studies in a mouse model. VLPs proved to be highly immunogenic and induced the production of high levels of neutralizing antibodies. In a challenge experiment, immunization with VLPs provided full protection from lethal TBE in mice. Thus, we suggest that Leishmania-derived VLPs may be a good candidate for a safe alternative human vaccine with high efficiency of production. Moreover, this potential vaccine candidate may constitute a low-cost candidate for veterinary use.

AddaVax-Adjuvanted H5N8 Inactivated Vaccine Induces Robust Humoral Immune Response against Different Clades of H5 Viruses.

Since some cases of human infections with H5N8 avian influenza virus have been reported and caused great concern in recent years, it is important to develop an effective vaccine for human use to prevent a potential H5N8 pandemic. In the present study, a vaccine candidate virus based on newly human-infected A/Astrakhan/3212/2020 H5N8 virus was constructed by reverse genetics (RG) technology. The immunogenicity of H5N8 whole virion inactivated vaccine was evaluated by various doses of vaccine antigen formulated with squalene-based adjuvant (AddaVax), aluminum hydroxide (Al(OH)3) or without adjuvant in mice. The results showed AddaVax-adjuvanted H5N8 inactivated vaccine could stimulate the mice to produce a stronger protective immune response with higher titers of IgG antibodies, hemagglutination inhibition (HI), neuraminidase inhibition (NI) and microneutralization (MN) antibodies than vaccine formulations with Al(OH)3 adjuvant or without adjuvant, and achieve a dose-sparing effect. Moreover, the AddaVax-adjuvanted formulation also exhibited potent cross-reactive response in HI antibodies against different clades of H5 viruses. A significant correlation and a curve fitting among HI, NI and MN were found by the correlation analysis to predict the protective effect of the vaccine. With these findings, our study demonstrates that AddaVax adjuvant can enhance the immunogenicity of H5N8 inactivated vaccine remarkably, and proposes an effective strategy for dealing with a potential H5N8 virus pandemic.

Cellular Affinity of Particle-Stabilized Emulsion to Boost Antigen Internalization.

The cellular affinity of micro-/nanoparticles is the precondition for cellular recognition, cellular uptake, and activation, which are essential for drug delivery and immune response. The present study stemmed from the observation that the effects of charge, size, and shape of solid particles on cell affinity are usually considered, but we seldom realize the essential role of softness, dynamic restructuring phenomenon, and complex interface interaction in cellular affinity. Here, we developed poly-lactic-co-glycolic acid (PLGA) nanoparticle-stabilized Pickering emulsion (PNPE) that overcame the shortcomings of rigid forms and simulated the flexibility and fluidity of pathogens. A method was set up to test the affinity of PNPE to cell surfaces and elaborate on the subsequent internalization by immune cells. The affinity of PNPE to bio-mimetic extracellular vesicles (bEVs)-the replacement for bone marrow dendritic cells (BMDCs)-was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D), which allowed real-time monitoring of cell-emulsion adhesion. Subsequently, the PNPE was used to deliver the antigen (ovalbumin, OVA) and the uptake of the antigens by BMDCs was observed using confocal laser scanning microscope (CLSM). Representative results showed that the PNPE immediately decreased frequency (ΔF) when it encountered the bEVs, indicating rapid adhesion and high affinity of the PNPE to the BMDCs. PNPE showed significantly stronger binding to the cell membrane than PLGA microparticles (PMPs) and AddaVax adjuvant (denoted as surfactant-stabilized nano-emulsion [SSE]). Furthermore, owing to the enhanced cellular affinity to the immunocytes through dynamic curvature changes and lateral diffusions, antigen uptake was subsequently boosted compared with PMPs and SSE. This protocol provides insights for designing novel formulations with high cell affinity and efficient antigen internalization, providing a platform for the development of efficient vaccines.

Formulation of an ovarian cancer vaccine with the squalene-based AddaVax adjuvant inhibits the growth of murine epithelial ovarian carcinomas.

PurposeEpithelial ovarian carcinoma (EOC) is the most lethal of all human gynecologic malignancies. We previously reported that vaccination of female mice with the extracellular domain of anti-Müllerian hormone receptor II (AMHR2-ED) in complete Freund’s adjuvant (CFA) generates AMHR2-ED specific immunoglobulin G (IgG) that provides prevention and therapy against murine EOCs. Although CFA is the “gold standard” adjuvant in animal studies, it is not approved for human use because it often induces painful granulomas and abscesses. Thus, the objective of this study is to identify an alternative adjuvant to CFA for use in our ovarian cancer vaccine clinical trials.Materials and MethodsBecause it has been used successfully without serious adverse effects in numerous human clinical trials, we selected the IgG-inducing squalene-based adjuvant, AddaVax™, for evaluation of its ability to facilitate vaccine-induced prevention and treatment of EOC in mice. To this end, we immunized female C57BL/6 mice with recombinant mouse AMHR2-ED emulsified with either AddaVax or CFA as adjuvant and compared the results.ResultsWe found that formulation of the AMHR2-ED vaccine with AddaVax adjuvant induced high serum titers of IgG and significant inhibition of EOC growth with significantly enhanced overall survival of mice using both prevention and therapeutic protocols. These results were compared favorably with results obtained using CFA as an adjuvant in the AMHR2-ED vaccine.ConclusionOur data indicate that the AMHR2-ED vaccine formulated with AddaVax may be used in human clinical trials and thereby serve as a novel and effective way to control human EOC.

Potentiation of Recombinant NP and M1-Induced Cellular Immune Responses and Protection by Physical Radiofrequency Adjuvant.

Nucleoprotein (NP) and matrix protein 1 (M1) are highly conserved among influenza A viruses and have been attractive targets to develop vaccines to elicit cross-reactive cytotoxic T lymphocytes (CTLs). Yet, external antigens are often presented on major histocompatibility complex class II molecules and elicit humoral immune responses. In this study, we present a physical radiofrequency adjuvant (RFA) to assist recombinant NP and M1 to elicit potent CTL responses. We found recombinant NP/M1 immunization in the presence of RFA could elicit potent anti-NP CTLs and confer significant protection against homologous viral challenges, while NP/M1 immunization alone failed to elicit significant CTL responses or confer significant protection. Interestingly, RFA failed to elicit potent anti-M1 CTL responses or anti-NP or anti-M1 antibody responses. Different from RFA, AddaVax adjuvant was found to significantly increase NP-specific antibody responses but not CTLs. NP/M1 immunization in the presence of RFA or AddaVax similarly reduced body weight loss, while only the former significantly increased the survival. We further found NP/M1 immunization in the presence of RFA did not significantly increase serum IL-6 release (a systemic inflammatory mediator) and rather reduced serum IL-6 release after boost immunization. NP/M1 immunization in the presence of RFA did not induce significant local reactions or increase body temperature of mice. The high potency and safety strongly support further development of RFA-based recombinant NP/M1 vaccine to elicit cross-protective immunity.

A chemokine-fusion vaccine targeting immature dendritic cells elicits elevated antibody responses to malaria sporozoites in infant macaques

Infants and young children are the groups at greatest risk for severe disease resulting from Plasmodium falciparum infection. We previously demonstrated in mice that a protein vaccine composed of the chemokine macrophage inflammatory protein 3α genetically fused to the minimally truncated circumsporozoite protein of P. falciparum (MCSP) elicits high concentrations of specific antibody and significant reduction of liver sporozoite load in a mouse model system. In the current study, a squalene based adjuvant (AddaVax, InvivoGen, San Diego, Ca) equivalent to the clinically approved MF59 (Seqiris, Maidenhead, UK) elicited greater antibody responses in mice than the previously employed adjuvant polyinosinic:polycytidylic acid, ((poly(I:C), InvivoGen, San Diego, Ca) and the clinically approved Aluminum hydroxide gel (Alum, Invivogen, San Diego, Ca) adjuvant. Use of the AddaVax adjuvant also expanded the range of IgG subtypes elicited by mouse vaccination. Sera passively transferred into mice from MCSP/AddaVax immunized 1 and 6 month old macaques significantly reduced liver sporozoite load upon sporozoite challenge. Protective antibody concentrations attained by passive transfer in the mice were equivalent to those observed in infant macaques 18 weeks after the final immunization. The efficacy of this vaccine in a relevant non-human primate model indicates its potential usefulness for the analogous high risk human population.

Development of high-growth influenza H7N9 prepandemic candidate vaccine viruses in suspension MDCK cells

BackgroundInfluenza vaccine manufacturers traditionally use egg-derived candidate vaccine viruses (CVVs) to produce high-yield influenza viruses for seasonal or pandemic vaccines; however, these egg-derived CVVs need an adaptation process for the virus to grow in mammalian cells. The low yields of cell-based manufacturing systems using egg-derived CVVs remain an unsolved issue. This study aimed to develop high-growth cell-derived CVVs for MDCK cell-based vaccine manufacturing platforms.MethodsFour H7N9 CVVs were generated in characterized Vero and adherent MDCK (aMDCK) cells. Furthermore, reassortant viruses were amplified in adherent MDCK (aMDCK) cells with certification, and their growth characteristics were detected in aMDCK cells and new suspension MDCK (sMDCK) cells. Finally, the plaque-forming ability, biosafety, and immunogenicity of H7N9 reassortant viruses were evaluated.ResultsThe HA titers of these CVVs produced in proprietary suspension MDCK (sMDCK) cells and chicken embryos were 2- to 8-fold higher than those in aMDCK cells. All H7N9 CVVs showed attenuated characteristics by trypsin-dependent plaque assay and chicken embryo lethality test. The alum-adjuvanted NHRI-RG5 (derived from the fifth wave H7N9 virus A/Guangdong/SP440/2017) vaccine had the highest immunogenicity and cross-reactivity among the four H7N9 CVVs. Finally, we found that AddaVax adjuvant improved the cross-reactivity of low pathogenic H7N9 virus against highly pathogenic H7N9 viruses.ConclusionsOur study indicates that cell-derived H7N9 CVVs possessed high growth rate in new sMDCK cells and low pathogenicity in chicken embryo, and that CVVs generated by this platform are also suitable for both cell- and egg-based prepandemic vaccine production.

Design and assessment of TRAP-CSP fusion antigens as effective malaria vaccines.

The circumsporozoite protein (CSP) and thrombospondin-related adhesion protein (TRAP) are major targets for pre-erythrocytic malaria vaccine development. However, the CSP-based vaccine RTS,S provides only marginal protection, highlighting the need for innovative vaccine design and development. Here we design and characterize expression and folding of P. berghei (Pb) and P. falciparum (Pf) TRAP-CSP fusion proteins, and evaluate immunogenicity and sterilizing immunity in mice. TRAP N-terminal domains were fused to the CSP C-terminal αTSR domain with or without the CSP repeat region, expressed in mammalian cells, and evaluated with or without N-glycan shaving. Pb and Pf fusions were each expressed substantially better than the TRAP or CSP components alone; furthermore, the fusions but not the CSP component could be purified to homogeneity and were well folded and monomeric. As yields of TRAP and CSP fragments were insufficient, we immunized BALB/c mice with Pb TRAP-CSP fusions in AddaVax adjuvant and tested the effects of absence or presence of the CSP repeats and absence or presence of high mannose N-glycans on total antibody titer and protection from infection by mosquito bite both 2.5 months and 6 months after the last immunization. Fusions containing the repeats were completely protective against challenge and re-challenge, while those lacking repeats were significantly less effective. These results correlated with higher total antibody titers when repeats were present. Our results show that TRAP-CSP fusions increase protein antigen production, have the potential to yield effective vaccines, and also guide design of effective proteins that can be encoded by nucleic acid-based and virally vectored vaccines.

Advancing Freund's and AddaVax Adjuvant Regimens Using CpG Oligodeoxynucleotides.

Adjuvant can play an important role in vaccine formulation by aiding in the development of a robust immune response. In our hybridoma development work, we typically use both Freund's and AddaVax™ adjuvant regimens for mouse immunizations. While we have repeatedly shown success with our protocols, we continually seek to improve upon the titer and affinity of the serum antibody response. To that end, we evaluated the use of CpG oligodeoxynucleotides (CpG-DNA), a B cell stimulant, in our adjuvant regimens. Mice were immunized using our standard Freund's protocol (Adjulite Complete Freund's Adjuvant for the primary immunization followed by Adjulite Incomplete Freund's Adjuvant (AIFA) for all additional immunizations) or a test protocol using AIFA supplemented with CpG-DNA for all immunizations. A second group of mice were immunized with antigen emulsified in AddaVax adjuvant alone or AddaVax supplemented with CpG-DNA. Our results show a trend toward a higher titer response when CpG-DNA was used with either adjuvant. In addition, AIFA+CpG-DNA mice trended toward a higher relative affinity versus mice immunized using our standard Freund's methodology. Additional antigens will need to be studied to determine whether these observations are limited to the proteins (antigens) studied or whether this is a generalized response to any immunogen.

Augmentation of vaccine-induced humoral and cellular immunity by a physical radiofrequency adjuvant

Protein/subunit vaccines often require external adjuvants to induce protective immunity. Due to the safety concern of chemical adjuvants, physical adjuvants were recently explored to boost vaccination. Physical adjuvants use physical energies rather than chemicals to stimulate tissue stress and endogenous danger signal release to boost vaccination. Here we present the safety and potency of non-invasive radiofrequency treatment to boost intradermal vaccination in murine models. We show non-invasive radiofrequency can increase protein antigen-induced humoral and cellular immune responses with adjuvant effects comparable to widely used chemical adjuvants. Radiofrequency adjuvant can also safely boost pandemic 2009 H1N1 influenza vaccination with adjuvant effects comparable to MF59-like AddaVax adjuvant. We find radiofrequency adjuvant induces heat shock protein 70 (HSP70) release and activates MyD88 to mediate the adjuvant effects. Physical radiofrequency can potentially be a safe and potent adjuvant to augment protein/subunit vaccine-induced humoral and cellular immune responses.

A novel multi-peptide subunit vaccine admixed with AddaVax adjuvant produces significant immunogenicity and protection against Proteus mirabilis urinary tract infection in mice model

Proteus mirabilis is a common pathogen in urinary tract infections (UTIs). There is no vaccine against P. mirabilis, thus a novel multi-peptide vaccine of MrpA, UcaA and Pta factors of P. mirabilis we designed and a mice model was used to evaluate its efficacy in combination with AddaVax adjuvant. According to the bioinformatics studies, 7 fragments of MrpA (31–75, 112–146), UcaA (68–117, 132–156) and Pta (210–265, 340–400, 496–570) with B and T cell epitope regions were selected for fusion construction. Mice subcutaneously vaccinated with the fusion MrpA.Pta.UcaA induced a significant increase in serum and mucosal IgG and IgA responses. The fusion also showed a significant induction in cellular responses (Th1 and Th2). The addition of AddaVax to fusion and the mixture of MrpA, UcaA, and Pta (MUP) improved the humoral and cellular responses, especially the IgG2a and IFN-γ (Th1 responses) levels. Fusion with and without AddaVax and MUP + AddaVax could maintain significant humoral responses until 6 months after the first vaccine dose. All vaccine combinations with and without adjuvant showed high effectiveness in the protection of the bladder and kidney against experimental UTI; this could be attributed to the significant humoral and cellular responses. The present study suggests that the AddaVax-based vaccine formulations especially the fusion Pta.MrpA.UcaA admixed with AddaVax as potential vaccine candidates for protection against P. mirabilis. Furthermore, AddaVax could be considered as an effective adjuvant in designing other vaccines against UTI pathogens.

Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen

Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 2040 patients and caused 712 deaths since its first appearance in 2012, yet neither pathogen-specific therapeutics nor approved vaccines are available. To address this need, we are developing a subunit recombinant protein vaccine comprising residues 377–588 of the MERS-CoV spike protein receptor-binding domain (RBD), which, when formulated with the AddaVax adjuvant, it induces a significant neutralizing antibody response and protection against MERS-CoV challenge in vaccinated animals. To prepare for the manufacture and first-in-human testing of the vaccine, we have developed a process to stably produce the recombinant MERS S377-588 protein in Chinese hamster ovary (CHO) cells. To accomplish this, we transfected an adherent dihydrofolate reductase-deficient CHO cell line (adCHO) with a plasmid encoding S377-588 fused with the human IgG Fc fragment (S377-588-Fc). We then demonstrated the interleukin-2 signal peptide-directed secretion of the recombinant protein into extracellular milieu. Using a gradually increasing methotrexate (MTX) concentration to 5 μM, we increased protein yield by a factor of 40. The adCHO-expressed S377-588-Fc recombinant protein demonstrated functionality and binding specificity identical to those of the protein from transiently transfected HEK293T cells. In addition, hCD26/dipeptidyl peptidase-4 (DPP4) transgenic mice vaccinated with AddaVax-adjuvanted S377-588-Fc could produce neutralizing antibodies against MERS-CoV and survived for at least 21 days after challenge with live MERS-CoV with no evidence of immunological toxicity or eosinophilic immune enhancement. To prepare for large scale-manufacture of the vaccine antigen, we have further developed a high-yield monoclonal suspension CHO cell line.

AddaVax vs. Freund’s adjuvant use for animal immunization in hybridoma development

Abstract Use of a potent adjuvant is essential for stimulating a strong, antigen specific response from immunized animals. While Freund’s continues as the gold standard for use in research animals we continuously evaluate available adjuvants as part of our improvement efforts in developing mouse and rabbit hybridomas. We recently evaluated AddaVax adjuvant for use in developing a high titer, high affinity immune response. Animals were immunized with peptides or recombinant proteins emulsified with Adjulite Freund’s adjuvant or AddaVax adjuvant. Sera samples were collected following the final immunization and the antibody evaluated for titer and relative affinity. Results show that the average serum antibody titer and relative affinity for AddaVax immunized mice were equivalent to or better than that from Freund’s mice for 3/7 peptides and 5/7 recombinant proteins. Data from two proteins also suggest AddaVax animals may show binding to antigenic epitopes not recognized by Freund’s animals. Disappointingly, rabbits immunized using AddaVax adjuvant consistently developed a much weaker response vs. Freund’s. Based on results generated, we have modified our protocols to include the use of Freund’s and AddaVax when initiating new hybridoma development projects in mice.

Carbohydrate fatty acid monosulphate esters are safe and effective adjuvants for humoral responses

Carbohydrate fatty acid sulphate esters (CFASEs) formulated in a squalane-in-water emulsion are effective adjuvants for humoral responses to a wide range of antigens in various animal species but rise in body temperature and local reactions albeit mild or minimal hampers application in humans. In rabbits, body temperature increased 1°C one day after intramuscular (IM) injection, which returned to normal during the next day. The effect increased with increasing dose of CFASE but not with the number of injections (up to 5). Antigen enhanced the rise in body temperature after booster immunization (P<0.01) but not after priming. Synthetic CFASEs are mixtures of derivatives containing no sulphate, one or multiple sulphate groups and the monosulphate derivatives (CMS) were isolated, incorporated in a squalane in-water emulsion and investigated. In contrast to CFASE, CMS adjuvant did not generate rise in body temperature or local reactions in rabbits immunized with a purified, recombinant malaria chimeric antigen R0.10C. In comparison to alum, CMS adjuvant revealed approximately 30-fold higher antibody titres after the first and >100-fold after the second immunization. In ferrets immunized with 7.5μg of inactivated influenza virus A/H7N9, CMS adjuvant gave 100-fold increase in HAI antibody titres after the first and 25-fold after the second immunisation, which were 10–20-fold higher than with the MF59-like AddaVax adjuvant. In both models, a single immunisation with CMS adjuvant revealed similar or higher titres than two immunisations with either benchmark, without detectable systemic and local adverse effects. Despite striking chemical similarities with monophospholipid A (MPL), CMS adjuvant did not activate human TLR4 expressed on HEK cells. We concluded that the synthetic CMS adjuvant is a promising candidate for poor immunogens and single-shot vaccines and that rise in body temperature, local reactions or activation of TLR4 is not a pre-requisite for high adjuvanticity.