The Matrix-M™ adjuvant: A critical component of vaccines for the 21st century.

Matrix-M™ adjuvant is a key component of several novel vaccine candidates. The Matrix-M adjuvant consists of two distinct fractions of saponins purified from the Quillaja saponaria Molina tree, combined with cholesterol and phospholipids to form 40-nm open cage-like nanoparticles, achieving potent adjuvanticity with a favorable safety profile. Matrix-M induces early activation of innate immune cells at the injection site and in the draining lymph nodes. This translates into improved magnitude and quality of the antibody response to the antigen, broadened epitope recognition, and the induction of a Th1-dominant immune response. Matrix-M-adjuvanted vaccines have a favorable safety profile and are well tolerated in clinical trials. In this review, we discuss the latest findings on the mechanisms of action, efficacy, and safety of Matrix-M adjuvant and other saponin-based adjuvants, with a focus on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subunit vaccine candidate NVX-CoV2373 developed to prevent coronavirus disease 2019 (COVID-19).

SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 immunogenicity in baboons and protection in mice.

The COVID-19 pandemic continues to spread throughout the world with an urgent need for a safe and protective vaccine to effectuate herd protection and control the spread of SARS-CoV-2. Here, we report the development of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) from the full-length spike (S) protein that is stable in the prefusion conformation. NVX-CoV2373 S form 27.2-nm nanoparticles that are thermostable and bind with high affinity to the human angiotensin-converting enzyme 2 (hACE2) receptor. In mice, low-dose NVX-CoV2373 with saponin-based Matrix-M adjuvant elicit high titer anti-S IgG that blocks hACE2 receptor binding, neutralize virus, and protects against SARS-CoV-2 challenge with no evidence of vaccine-associated enhanced respiratory disease. NVX-CoV2373 also elicits multifunctional CD4+ and CD8+ T cells, CD4+ follicular helper T cells (Tfh), and antigen-specific germinal center (GC) B cells in the spleen. In baboons, low-dose levels of NVX-CoV2373 with Matrix-M was also highly immunogenic and elicited high titer anti-S antibodies and functional antibodies that block S-protein binding to hACE2 and neutralize virus infection and antigen-specific T cells. These results support the ongoing phase 1/2 clinical evaluation of the safety and immunogenicity of NVX-CoV2373 with Matrix-M (NCT04368988).

Matrix-M™ adjuvant enhances immunogenicity of both protein- and modified vaccinia virus Ankara-based influenza vaccines in mice.

Influenza viruses continuously circulate in the human population and escape recognition by virus neutralizing antibodies induced by prior infection or vaccination through accumulation of mutations in the surface proteins hemagglutinin (HA) and neuraminidase (NA). Various strategies to develop a vaccine that provides broad protection against different influenza A viruses are under investigation, including use of recombinant (r) viral vectors and adjuvants. The replication-deficient modified vaccinia virus Ankara (MVA) is a promising vaccine vector that efficiently induces B and T cell responses specific for the antigen of interest. It is assumed that live vaccine vectors do not require an adjuvant to be immunogenic as the vector already mediates recruitment and activation of immune cells. To address this topic, BALB/c mice were vaccinated with either protein- or rMVA-based HA influenza vaccines, formulated with or without the saponin-based Matrix-M™ adjuvant. Co-formulation with Matrix-M significantly increased HA vaccine immunogenicity, resulting in antigen-specific humoral and cellular immune responses comparable to those induced by unadjuvanted rMVA-HA. Of special interest, rMVA-HA immunogenicity was also enhanced by addition of Matrix-M, demonstrated by enhanced HA inhibition antibody titres and cellular immune responses. Matrix-M added to either protein- or rMVA-based HA vaccines mediated recruitment and activation of antigen-presenting cells and lymphocytes to the draining lymph node 24 and 48 h post-vaccination. Taken together, these results suggest that adjuvants can be used not only with protein-based vaccines but also in combination with rMVA to increase vaccine immunogenicity, which may be a step forward to generate new and more effective influenza vaccines.

Matrix-M adjuvant enhances antibody, cellular and protective immune responses of a Zaire Ebola/Makona virus glycoprotein (GP) nanoparticle vaccine in mice.

Ebola virus (EBOV) causes severe hemorrhagic fever for which there is no approved treatment or preventive vaccine. Immunological correlates of protective immunity against EBOV disease are not well understood. However, non-human primate studies have associated protection of experimental vaccines with binding and neutralizing antibodies to the EBOV glycoprotein (GP) as well as EBOV GP-specific CD4(+) and CD8(+) T cells. In this report a full length, unmodified Zaire EBOV GP gene from the 2014 EBOV Makona strain (EBOV/Mak) was cloned into a baculovirus vector. Recombinant EBOV/Mak GP was produced in Sf9 insect cells as glycosylated trimers and, when purified, formed spherical 30-40 nm particles. In mice, EBOV/Mak GP co-administered with the saponin adjuvant Matrix-M was significantly more immunogenic, as measured by virus neutralization titers and anti-EBOV/Mak GP IgG as compared to immunization with AlPO4 adjuvanted or non-adjuvanted EBOV/Mak GP. Similarly, antigen specific T cells secreting IFN-γ were induced most prominently by EBOV/Mak GP with Matrix-M. Matrix-M also enhanced the frequency of antigen-specific germinal center B cells and follicular helper T (TFH) cells in the spleen in a dose-dependent manner. Immunization with EBOV/Mak GP with Matrix-M was 100% protective in a lethal viral challenge murine model; whereas no protection was observed with the AlPO4 adjuvant and only 10% (1/10) mice were protected in the EBOV/Mak GP antigen alone group. Matrix-M adjuvanted vaccine induced a rapid onset of specific IgG and neutralizing antibodies, increased frequency of multifunctional CD4+ and CD8(+) T cells, specific TFH cells, germinal center B cells, and persistence of EBOV GP-specific plasma B cells in the bone marrow. Taken together, the addition of Matrix-M adjuvant to the EBOV/Mak GP nanoparticles enhanced both B and T-cell immune stimulation which may be critical for an Ebola subunit vaccine with broad and long lasting protective immunity.

Vaccine-induced protection of rhesus macaques against plasma viremia after intradermal infection with a European lineage 1 strain of West Nile virus.

The mosquito-borne West Nile virus (WNV) causes human and animal disease with outbreaks in several parts of the world including North America, the Mediterranean countries, Central and East Europe, the Middle East, and Africa. Particularly in elderly people and individuals with an impaired immune system, infection with WNV can progress into a serious neuroinvasive disease. Currently, no treatment or vaccine is available to protect humans against infection or disease. The goal of this study was to develop a WNV-vaccine that is safe to use in these high-risk human target populations. We performed a vaccine efficacy study in non-human primates using the contemporary, pathogenic European WNV genotype 1a challenge strain, WNV-Ita09. Two vaccine strategies were evaluated in rhesus macaques (Macaca mulatta) using recombinant soluble WNV envelope (E) ectodomain adjuvanted with Matrix-M, either with or without DNA priming. The DNA priming immunization was performed with WNV-DermaVir nanoparticles. Both vaccination strategies successfully induced humoral and cellular immune responses that completely protected the macaques against the development of viremia. In addition, the vaccine was well tolerated by all animals. Overall, The WNV E protein adjuvanted with Matrix-M is a promising vaccine candidate for a non-infectious WNV vaccine for use in humans, including at-risk populations.

Matrix-M™ adjuvanted envelope protein vaccine protects against lethal lineage 1 and 2 West Nile virus infection in mice.

West Nile virus (WNV) is a mosquito-transmitted flavivirus and an emerging pathogen in many parts of the world. In the elderly and immunosuppressed, infection can progress rapidly to debilitating and sometimes fatal neuroinvasive disease. Currently, no WNV vaccine is approved for use in humans. As there have been several recent outbreaks in the United States and Europe, there is an increasing need for a human WNV vaccine. In this study, we formulated the ectodomain of a recombinant WNV envelope (E) protein with the particulate saponin-based adjuvant Matrix-M™ and studied the antigen-specific immune responses in mice. Animals immunized with Matrix-M™ formulated E protein developed higher serum IgG1 and IgG2a and neutralizing antibody titers at antigen doses ranging from 0.5 to 10 µg compared to those immunized with 3 or 10 µg of E alone, E adjuvanted with 1% Alum, or with the inactivated virion veterinary vaccine, Duvaxyn(®) WNV. This phenotype was accompanied by strong cellular recall responses as splenocytes from mice immunized with Matrix-M™ formulated vaccine produced high levels of Th1 and Th2 cytokines. Addition of Matrix-M™ prolonged the duration of the immune response, as elevated humoral and cellular responses were maintained for more than 200 days. Importantly, mice vaccinated with Matrix-M™ formulated E protein were protected from lethal challenge with both lineage 1 and 2 WNV strains. In summary, Matrix-M™ adjuvanted E protein elicited potent and durable immune responses that prevented lethal WNV infection, and thus is a promising vaccine candidate for humans.

Immune enhancing properties of the novel Matrix-M™ adjuvant leads to potentiated immune responses to an influenza vaccine in mice.

The novel saponin based adjuvant Matrix-M™ was recently used in a Phase I study of seasonal influenza in elderly. The present study is a pre-clinical evaluation of the efficacy and mode-of-action of Matrix-M™ formulated influenza vaccine in mice. A manuscript on safety profile and immunogenicity in elderly humans is under preparation. We have previously shown that subcutaneous injections of Matrix-M™, without coformulated antigen, results in a dose-dependent recruitment of leukocytes to draining lymph nodes (dLNs). Herein we compared the mode of action of Matrix-M™ with Alum, FCA and AS03 alone or formulated with influenza split virion antigen injected intramuscularly. The elicited responses in dLNs and spleen were investigated 48h later. Matrix-M™ was particularly efficient in activation of central innate immune cells such as neutrophils, DCs and macrophages compared to the other adjuvants analyzed. Moreover, the adjuvant influence on the recall immune response to influenza antigen was studied by in vitro re-stimulation of splenocytes from mice immunized with influenza antigen adjuvanted with Matrix-M™, Alum or AS03. Splenocytes from mice immunized with influenza antigen and Matrix-M™ produced both Th1 and Th2 cytokines upon re-stimulation. This response was significantly stronger than that induced by the other adjuvants studied. Interestingly, increased levels of the neutrophil chemoattractant KC were produced by antigen stimulated splenocytes from mice immunized with Matrix-M™ adjuvanted vaccine, which is in agreement with the increase of neutrophils into dLNs and spleen after Matrix-M™ injection. Furthermore, influenza antigen adjuvanted with Matrix-M™ induced significantly higher antigen-specific IgG1 and IgG2a responses compared to antigen alone. In conclusion, adjuvant Matrix-M™ activates the innate immune system without antigen present. This activation may explain the enhanced immunity to influenza seen with Matrix-M™ adjuvant. Despite this potent immune activation mediated by Matrix-M™, GLP-toxicity studies and clinical data suggest that Matrix-M™ adjuvant has a mild to moderate safety profile.

Immunization of macaques with soluble HIV type 1 and influenza virus envelope glycoproteins results in a similarly rapid contraction of peripheral B-cell responses after boosting.

The envelope glycoproteins (Env) represent a critical component of a successful antibody-mediated human immunodeficiency virus type 1 (HIV-1) vaccine. However, immunization with soluble Env was reported to induce short-lived antibody responses, suggesting that Env has unusual immunogenic properties. Here, we directly compared the magnitude and durability of B-cell responses induced by HIV-1 Env and an unrelated soluble viral protein, influenza virus hemagglutinin (HA), in simultaneously inoculated macaques. We demonstrate robust peak responses followed by rapid contraction of circulating antibody and memory B cells for both antigens, suggesting that short-lived responses are not unique to HIV-1 Env but may be a common feature of soluble protein vaccines.

Matrix-M™ adjuvant induces local recruitment, activation and maturation of central immune cells in absence of antigen.

Saponin-based adjuvants are widely used to enhance humoral and cellular immune responses towards vaccine antigens, although it is not yet completely known how they mediate their stimulatory effects. The aim of this study was to elucidate the mechanism of action of adjuvant Matrix-M™ without antigen and Alum was used as reference adjuvant. Adjuvant Matrix-M™ is comprised of 40 nm nanoparticles composed of Quillaja saponins, cholesterol and phospholipid. BALB/c mice were subcutaneously injected once with, 3, 12 or 30 µg of Matrix-M™, resulting in recruitment of leukocytes to draining lymph nodes (dLNs) and spleen 48 h post treatment. Flow cytometry analysis identified CD11b(+) Gr-1(high) granulocytes as the cell population increasing most in dLNs and spleen. Additionally, dendritic cells, F4/80(int) cells, T-, B- and NK-cells were recruited to dLNs and in spleen the number of F4/80(int) cells, and to some extent, B cells and dendritic cells, increased. Elevated levels of early activation marker CD69 were detected on T-, B- and NK-cells, CD11b(+) Gr-1(high) cells, F4/80(int) cells and dendritic cells in dLNs. In spleen CD69 was mainly up-regulated on NK cells. B cells and dendritic cells in dLNs and spleen showed an increased expression of the co-stimulatory molecule CD86 and dendritic cells in dLNs expressed elevated levels of MHC class II. The high-dose (30 µg) of Matrix-M™ induced detectable serum levels of IL-6 and MIP-1ß 4 h post administration, most likely representing spillover of locally produced cytokines. A lesser increase of IL-6 in serum after administration of 12 µg Matrix-M™ was also observed. In conclusion, early immunostimulatory properties were demonstrated by Matrix-M™ alone, as therapeutic doses resulted in a local transient immune response with recruitment and activation of central immune cells to dLNs. These effects may play a role in enhancing uptake and presentation of vaccine antigens to elicit a competent immune response.

Effects of chronic stress on the immune response to oral human serum albumin-conjugated starch microparticles in rats.

Uptake of antigens and bacteria over the follicle-associated epithelium (FAE) is increased after chronic psychological stress. We investigated whether stress affects the immune response to particle-conjugated antigens taken up via the FAE. Rats were submitted to two 10-day periods of water avoidance stress and orally immunized during these periods. Stressed immunized rats displayed altered cell populations and a Th1-skewed immune response within the lymphoid follicles, together with enhanced delayed-type hypersensitivity. We conclude that chronic stress affects the cell-mediated immune response after oral immunization, which may have implications for the understanding of allergic and autoimmune diseases and development of oral vaccines.

Use of an oral diphtheria vaccine in human.

Starch microparticles have been shown to be effective as a particulate adjuvant carrier in oral vaccination. In mice, formulations with bacterial antigens have elicited both systemic and mucosal immune responses providing protection upon challenge with live bacteria. A vaccine formulation with formaldehyde-treated diphtheria toxin cross-reacting material, CRM197, optimised in mice, was tested in healthy volunteers in a booster design. Specific antibodies as well as toxin-neutralising antibodies in a Vero cell analysis indicated that the vaccine was not effective in man. It is obvious that the longer transit time in the human GI tract and possible unfavourable distribution of Peyer's patches and M-cells necessary for the uptake of the starch particles require a more stable formulation. It is proposed that enteric coating of the particles or particles in a gastro-resistant capsule could be a more efficacious vaccine formulation.

Starch microparticles as a vaccine adjuvant: only uptake in Peyer's patches decides the profile of the immune response.

Starch microparticles, an effective adjuvant for oral vaccination in mice, are taken up over the follicle-associated epithelium (FAE) in Peyer's patches when human serum albumin is conjugated to the particles (HSAmp). When recombinant cholera toxin B subunit (rCTBmp) is conjugated, they are taken up over both the FAE and the villus epithelium. This study investigated the effects of the different targeting on the immune response by using particles with both HSA and rCTB (HSA/rCTBmp). The response induced after oral immunisation with this formulation in mice was compared with that obtained after administration of HSAmp, rCTBmp or both given concomitantly (HSAmp+rCTBmp) and after subcutaneous administration. Both the HSA- and rCTB-specific responses were followed quantitatively (as assessed by the anti-[IgM+IgG] level, the s-IgA response and the delayed-type hypersensitivity [DTH] response), or qualitatively (by the IgG subclass profile). After subcutaneous administration, the rCTB-specific IgM+IgG and DTH responses were lower after HSA/rCTBmp than after rCTBmp and the HSA-specific subclass ratio (IgG1/IgG2a+IgG2b) was lower with HSAmp+rCTBmp (but not with HSA/rCTBmp) compared to HSAmp. However, no quantitative and qualitative differences in the immune response after oral administration were detected when rCTB was added to HSAmp. The results indicate that only the uptake over the Peyer's patches decides the immune responses after oral administration and that the increased targeting to GM1 receptors of the villus epithelium does not affect the immune response. Moreover, the qualitative Th1/Th2-balance is controlled by the inherent properties of the antigens in the microparticles upon subcutaneous administration. Thus, the obtained information is important for designing oral microparticulate vaccines in order to obtain the wanted immune response.

Starch microparticles as vaccine adjuvant.

The demand for new vaccine adjuvants is well documented. New purified antigens from parasites, bacterial or viral pathogens, as well as recombinant subunit antigens and synthetic peptides, are often inherently weak immunogens; therefore, they need some kind of adjuvant to help initiate an immune response. In addition, there are very few adjuvants using the potential of the mucosal immune system, which may play an important role in the defence against air- and food-borne infections. Starch is a natural biocompatible and biodegradable polymer that is suitable for the production of various particulate adjuvant formulations, which can induce mucosal as well as systemic immune responses. This review gives an account of the different starch adjuvants used in immunisation studies. In particular, the properties of polyacryl starch microparticles as an oral vaccine adjuvant that induce protective immune responses in mice challenge experiments are summarised. In addition, a diphtheria booster vaccine has been proposed to be used to proving the concept in man and the possibilities to design an efficient vaccine formulation for human use are discussed.

Starch microparticles as oral vaccine adjuvant: antigen-dependent uptake in mouse intestinal mucosa.

An oral vaccine formulation comprised of starch microparticles with conjugated antigens is being developed. In this report we have examined the uptake of such microparticles by the intestinal mucosa and examined whether the conjugated antigen can influence the uptake. Two model antigens were used: recombinant cholera toxin B subunit (rCTB), which is known to bind to the ubiquitous GM1-receptor, and human serum albumin (HSA) which is not known to have any specific binding properties. The uptake was studied in mouse ligated intestinal loops into which the microparticles were injected. The intestinal loops were excised, fixed in ice-cold 95% ethanol. Entire specimens were mounted, exposed to fluorescence-labeled reagents staining the cytoskeleton, the particles and/or M cells and examined in a confocal laser-scanning microscope. A qualitative difference in the uptake of the rCTB- and HSA-conjugated microparticles was seen. The rCTB-conjugated microparticles were found both in villi and in the follicles of the Peyer's patches. HSA-conjugated microparticles could only be detected in the follicles of the Peyer's patches and not in villi. The rCTB conjugated to the microparticles did not lose its ability to bind the GM1-receptor, as shown with a GM1-ELISA, and the uptake of rCTB-conjugated microparticles in villi is most probably facilitated by the rCTB binding to the GM1-receptor. The qualitative difference in uptake could be of importance for the development of an immune response as the cytokine and chemokine microenvironment during antigen presentation will decide the differentiation of the immune response induced.

Starch microparticles as an adjuvant in immunisation: effect of route of administration on the immune response in mice.

This paper describes the effects on the development of an immune response by changing the route of administration of a new vaccine adjuvant, starch microparticles with human serum albumin (HSA) as a model antigen. The model vaccine was administered to mice by oral, subcutaneous and intramuscular routes in various combinations and both the local secretory immunoglobulin antibody (s-IgA) and systemic humoral and cellular (delayed-type hypersensitivity assay (DTH)) responses were followed. The only immunisation regimens inducing a significant s-IgA response were those incorporating oral booster doses. Oral and subcutaneous immunisations had similar effects on the Th1/Th2 balance, as indicated by the IgG subclass ratios and cytokine analyses. However, significant differences between oral and intramuscular immunisations were seen in the IgG subclass ratios. The Th2 influence was stronger after oral primary immunisation than after intramuscular primary immunisation, while oral boosters elicited a comparatively stronger Th1 response than intramuscular boosters. This result was also supported by the DTH analyses. Subcutaneous immunisation induced a stronger Th2 response than intramuscular immunisation, as indicated by subclass ratio and the IgE response. In conclusion, our results show that the profile of an immune response depends on the route of administration, which should be considered when developing new vaccines or new routes of administration.