COVID-19: Scientists in Australia have developed a method for the rapid synthesis of safe vaccines

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Scientists in Australia have developed a method for the rapid synthesis of safe vaccines, an approach that can be used to test vaccine strategies against novel pandemic pathogens such as SARS-CoV-2, the virus that causes COVID-19.

Led by Professor Richard Payne at the University of Sydney and Professor Warwick Britton at the Centenary Institute, the team has demonstrated application of the method with a new vaccine for use against tuberculosis (TB), which has generated a powerful protective immune response in mice.

Researchers are keen to develop the vaccine strategy further to assist in the rapid pre-clinical testing of new vaccines, particularly for respiratory illnesses.

“Tuberculosis infects 10 million and kills more than 1.4 million people every year,” said joint first author Dr. Anneliese Ashhurst from the University of Sydney. “Historically, it is the leading cause of death worldwide from a single infectious agent. So far, a TB vaccine that is highly effective and safe to use in all populations has eluded medical science.”

The only current vaccine for tuberculosis, the Bacille Calmette-Guerin vaccine, uses an injected live bacterium. It is effective in infants but has reduced effectiveness in adolescents and adults and poses significant health risks for immunocompromised patients, particularly for people living with HIV/AIDS.

Protein-based vaccines have been shown to be very safe, but they must be mixed with enhancers, or adjuvants, to make them effective, which is not straightforward.

Dr. Ashhurst said: “The challenge is to ensure that our immune cells see both the protein and adjuvant simultaneously. To overcome this difficulty, for the first time we have developed a method that synthesizes the protein with an attached adjuvant as a single molecule.”

The vaccine strategy and synthetic technology could be deployed to rapidly generate new vaccines for pre-clinical testing for a range of diseases, the researchers say, including the respiratory pathogen that causes COVID-19.

Their results are published today in the Proceedings of the National Academy of Sciences.

How it works

In order for vaccines to be effective, they need to stimulate behavior in protective T-cells that allows them to recognize the pathogen as an antigen, or foreign body. In the case of tuberculosis, our immune system needs to respond quickly to the bacteria that causes TB – Mycobacterium tuberculosis – to reduce infection in lungs.

Using the method developed by the Sydney scientists, an inhaled vaccine provides a low-dose immune-stimulating molecule – containing a synthesised bacterial protein attached directly to an adjuvant – to the immune cells in the lungs.

A major hurdle overcome by the scientists was the difficulty in fusing hydrophobic (water-repellent) adjuvants with a water-soluble protein antigen.

“We got around this problem of keeping hydrophobic and hydrophilic molecules together in a vaccine by developing a way to permanently bind the protein and adjuvant together as a single molecule using synthetic chemistry. Our approach overcomes the solubility problems faced by other methods,” said Professor Payne from the School of Chemistry and Deputy Director of the ARC Centre for Innovations in Peptide & Protein Science (CIPPS).

The team says that synthesizing an entire bacterial protein with attached adjuvant has not been achieved before.

Professor Britton from the Tuberculosis Research Program at the Centenary Institute said: “As well as providing a rapid method to develop a range of vaccines for pre-clinical testing, we expect that this pulmonary vaccination approach will be particularly beneficial for protecting against respiratory diseases.”

He said: “We hope that an inhaled vaccine for tuberculosis using a protein-based immunization will allow us to develop a universal and safe approach to combatting this deadly disease.”

The other major advantage with this method is that vaccines for a range of diseases can be developed rapidly and safely in the laboratory.

“We don’t need to grow the actual pathogen in the lab to make the vaccine,” said Dr. Ashhurst, who holds a joint position in the School of Chemistry and the School of Medical Sciences. “Using this new method, we can rapidly and safely synthesize highly pure vaccines in the lab and take them straight into animal models for pre-clinical testing.”


While vaccines were originally made from live attenuated microorganisms, modern biotechnology, through the use of genetic engineering, contributed to the development of vaccines containing highly purified recombinant antigens, which has been gradually replacing self-adjuvanted live attenuated and killed vaccine formulations.

This approach has successfully lowered reactogenicity rates as compared with live attenuated or killed vaccines. However, recombinant-antigen-based vaccines are often insufficiently immunogenic, especially in populations with distinct immunity, such as the very young, elderly, and chronically ill, highlighting the need for approaches to amplify protective vaccine responses.

Originally named on the Latin root adjuvare, to help or aid, adjuvants are defined as components which can enhance antigen-specific vaccine immunogenicity. Used only when needed, as vaccine formulations should be kept as simple as possible for development and regulatory purposes, adjuvants can be key for antigen/dose-sparing, broadening immunity to variable antigens and enhancing responses from vulnerable populations with weak immune responses.

Characterizing adjuvant mechanism of action (MOA) is important for effective translation and increasingly desirable from a regulatory and licensing perspective. Aluminum salts (alum) have been widely used to enhance humoral immunity, but their MOA is still under study [1–3]. Over the past 2 decades, a greater understanding of innate immunity, including identification of pattern recognition receptors (PRRs), has informed development of modern adjuvants [4,5].

Because the immunostimulatory effects of adjuvants can potentially induce undesirable reactogenicity, discovery and development must focus on yielding adjuvanted vaccines that are not only immunogenic, but also highly tolerable.

Herein, we comprehensively review current available adjuvants incorporated into modern vaccines, as well highlight several new classes under investigation, with a focus on MOA for enhancing immunogenicity as well as mechanisms that may impact safety, such as reactogenicity. To inform this review, we performed literature searches in PubMed with terms such as ‘adjuvant’ ‘vaccines’ AND/OR ‘safety’ AND/OR ‘reactogenicity’ in November 2019. Titles and abstracts of articles were screened by two authors (E.N. and O.L.), and those deemed most relevant, comprehensive and timely were reviewed.

OVERVIEW OF VACCINE ADJUVANTS
Alum is the most widely used vaccine adjuvant in history. Depot mechanisms were initially hypothesized to be the main MOA for alum’s adjuvanticity. However, alum injection sites can be excised shortly after injection with no impact on immunogenicity [6].

Several MOAs, both direct and indirect, are thought to contribute to alum-mediated enhancement of antibody production [7,8], including first, enhancement of antigen delivery to antigen presenting cells (APCs) such as macrophages and dendritic cells [9,10], partially through preferential binding of lipids on the surface of dendritic cells without alum itself being internalized by the cells [11]; second, triggering of innate immunity via inflammasome complexes inducing production of IL-1β, independent of Toll-like receptor (TLR) signaling [3]; and third, induction of cell death with consequent release of host cell DNA that can act as an endogenous adjuvant [2].

Alum has been also used as a component of combination adjuvantation systems wherein it is codelivered with other adjuvants such as TLR agonists (TLRAs) [7]. While alum has been the main adjuvant in vaccine formulations licensed for pediatric use in the United States, other adjuvants have been or are currently employed in licensed pediatric vaccines including live attenuated vaccines containing endogenous adjuvants (i.e., are ‘self-adjuvanted’) such as Bacille Calmette-Guérin (BCG), given at birth or early infancy in tuberculosis-endemic countries, that activates multiple PRRs including TLRs 2, 4, and 8 as well as the C-type lectin receptor Mincle [12,13], or exogenously added adjuvants such as the formerly used outer membrane protein-adjuvanted Hib vaccine that activates TLR2 [14] as well as human papilloma vaccine (Cervarix) containing monophosphoryl lipid a (MPLA), available to in the European Union and China for girls (staring at age 9 years) and adolescents and young adults (from 16 to 25 years of age) (Table 1).

Table 1
US Food and Drug Administration-approved vaccines containing novel adjuvants

Diverse water-in-oil emulsions were originally evaluated in human trials during the mid-20th century [15]. Water-in-oil emulsions, wherein water droplets are held within a continuous mineral oil phase, either containing killed Mycobacterium tuberculosis (Freund complete adjuvant) or not (Freund incomplete adjuvant) provided potent immunogenicity and were used in early influenza vaccines [16,17]. However, they demonstrated intolerable reactogenicity such as abscess and cyst formation at the site of injection [18]. Furthermore, instability of the antigen and lack of formulation reproducibility led to the consensus to avoid the use of water-in-oil for human prophylactic vaccines for infectious diseases, although some water-in-oil emulsions are still used as therapeutic vaccines for cancer [19,20].

Oil-in-water emulsions were developed as an alternative to water-in-oil emulsions and demonstrated significantly better reactogenicity profiles. The most common oil phase contained in vaccine formulations is squalene, a natural organic compound originally obtained for commercial purposes from shark liver oil and some plant sources. Since the introduction of MF59, that enhances both humoral and cell mediated Th1 and Th2 responses, oil-in-water emulsions have been routinely used in many seasonal and pandemic influenza vaccines for adults [21–23].

The MOA of MF59, a squalene-containing oil-in-water emulsion, appears to include enhancing antigen uptake of monocytes and dendritic cells [24]; and secretion of chemokines to create an ‘immunocompetent environment’ for enhanced antigen transportation to the draining lymph nodes [25]. AS03, an oil-in-water emulsion containing squalene, polysorbate 80 and α-tocopherol (a form of vitamin E), is a component of multiple influenza vaccines (e.g., Pandemrix, Arepanrix). Studied in mice, α-tocopherol in AS03 modulated cytokine and chemokine expression, increased antigen loading in monocytes, enhanced recruitment of granulocytes in draining lymph nodes, and enhanced antibody responses [26].

Saponins are triterpenoid molecules extracted from a variety of plants. Quil-A, which is a heterogeneous product extracted from the Chilean soapbark tree Quillaja saponaria, has been used in veterinary vaccinology since the 1950s. Although Quil-A had excessive reactogenicity for human use, its affinity for cholesterol had prompted development of immune stimulating complexes (ISCOMs) [27,28].

ISCOMs are spherical cage-like nanoparticles formed via self-assembly of a mixture of Quil-A, cholesterol, phospholipids, and antigens [29,30]. ISCOMs in the absence of an antigen (called ISCOMATRIX) can be mixed with an antigen of interest. ISCOMs stimulate enhanced cellular responses with lower antigen doses through enhanced antigen cross-presentation [31].

The saponin QS-21, a natural compound extracted from Q. saponaria, consists of a single saponin peak detectable by HPLC and is a component, together with MPLA, of the AS01B-adjuvanted zoster subunit vaccine (Shingrix) (Table 1) [32,33]. The combination adjuvant systems AS01 and AS02, components of the candidate RTS,S malaria vaccine, also contain both MPLA and QS21.

Although the exact MOA of QS-21 is not fully elucidated, the nanoparticulate nature of saponin/ISCOM formulations may lead to their preferential interaction and pore formation within cholesterol-rich dendritic cell membranes [4]. Furthermore, QS-21 elicits synergistic NLRP3-Asc-caspase-1-dependent IL-1β and IL-18 release in APCs when costimulated with MPLA [34].

The discovery of PRRs has accelerated discovery and development of PRR agonists as adjuvants. A number of these are now in clinical use or late preclinical stages of development. At the forefront is the 3′-deacylated monophosphoryl lipid A (MPLA), a TLR4A. TLR4 recognizes several pathogen-associated molecular patterns, including lipopolysaccharides (LPS) from the outer membrane of Gram-negative bacteria.

Although LPS have long been recognized as a potent adjuvant, its pyrogenic activity had limited its use in human [35]. MPLA is a detoxified form of the LPS from the bacterium Salmonella minnesota, with significantly lower reactogenicity (∼1000-fold lower), but robust adjuvanticity via Th1 polarization [4,36].

Synthetic single-stranded oligodeoxynucleotides (ODNs) containing unmethlyated cytosine phosphate guanine (CpG) motifs (CpG ODNs) found in bacterial DNA, have demonstrated adjuvant activity as a TLR9A [37]. CpG-ODN enhance antibody responses and enhance Th1-cell responses [38]. In humans, CpG motifs are recognized by TLR9 expressed on natural killer cells, B cells, and plasmacytoid dendritic cells but not myeloid dendritic cells and monocytes [39]. The licensed hepatitis B vaccine, Heplisav-B, indicated for use in adults at least 18 years, contains a TLR9A CpG adjuvant and, as compared with three doses of conventional alum adjuvanted hepatitis B vaccine, induces superior immunogenicity in older adults and the elderly (40–70 years of age) with only two doses [40,41].

Small molecule imidazoquinolines (IMQs) such as resiquimod (R848) are TLR7/8As [42]. These molecules activate human plasmacytoid dendritic cells and myeloid dendritic cells, enhancing expression of costimulatory molecules and production of type I IFN and IL-12 [43,44]. Although use of these small IMQ molecules is limited by their low molecular weight and rapid removal from the site of injection, several formulation methods, including covalent lipidation or incorporation into nanoparticles, enable use of these molecules as effective adjuvants in vivo[45]. Additional PRR agonists, including agonists of TLR3, TLR5, C-type lectin receptors, retinoic acid-inducible gene (RIG)-like receptors, and the stimulator of interferon genes are under evaluation as potential vaccine adjuvants [4,46,47].

Virosomes are enveloped virus-like particles that contain viral proteins in the liposomal membrane, and can act as adjuvants and carrier system for vaccinal antigens [48]. They are typically produced from reconstituted envelopes of influenza viruses, and enable robust and long-lasting immune responses with an excellent safety profile [49]. Virosomes have been licensed in vaccines against hepatitis A and pandemic influenza (Table 1), and also used in several clinical trials of malaria and hepatitis C vaccines.

Several additional types of adjuvants are under development. Proinflammatory cytokines such as granulocyte-macrophage colony-stimulating factor, IL-2, IL-12, and IL-15 were evaluated in vaccines against foot and mouth disease, hepatitis B, and HIV [50–53]. Diphtheria, tetanus, pertussis, and poliomyelitis vaccines containing calcium phosphate, in the form of hydroxyapatite nanoparticles with Th-polarizing cytokine inducing activity, have been licensed in France [54].

Combination adjuvantation systems can be a powerful approach to enhance immunogenicity [55]. As with any pharmacologic agents, adjuvant combinations can demonstrate additivity, antagonism or synergy [56]. Indeed, several of adjuvantation systems contained in licensed vaccines are comprised of combination systems such as AS01 (MPLA + QS21 in liposomes), AS02 (MPLA + QS21 in oil-in-water emulsion), AS03 (squalene + alpha-tocopherol in oil-in-water emulsion), and AS04 (MPL + alum) [57]. Of note, effects of at least some combination adjuvants may vary with age [58], though this has not been systematically studied.

ADJUVANT SAFETY
Although adjuvants are added to many vaccines for their immunostimulatory effects, they can potentially simultaneously induce undesirable reactogenicity, physical manifestations of the immunomodulatory and/or inflammatory response occurring within 72 h of vaccination [77▪▪,78▪].

Reactogenicity can be divided into local and systemic depending on the site of symptoms. Local reactogenicity includes erythema, swelling, pain, tenderness, or induration at the injection site, while systemic reactogenicity, often referred to as ‘flu-like’ symptoms, include chills, fever, fatigue, nausea, arthritis, myalgia, and headache.

Since the introduction of novel adjuvants, safety experience has been accumulated with their use in diverse vaccines as well as different target populations and settings. Although the safety profile of one adjuvanted vaccine in one target population cannot be extrapolated to other vaccines or populations, novel adjuvants showed no increase in serious adverse events so far. However, some vaccines with novel adjuvants had higher rates of local reactogenicity compared with their controls (placebo, nonadjuvanted, or alum-adjuvanted vaccines) [57,79▪].

A systematic review evaluating safety data in vaccine trials in adults at least 50 years, focused on AS01 (liposomal MPLA and saponin QS21), AS02 (oil-in-water with MPLA and QS21), AS03 (squalene-based adjuvant), and MF59 [80▪▪]. Rates of local pain were as high as 45.7–91.1% for AS01/AS02, 41.0–68.6% for AS03, and 0.7–64.8% for MF59, respectively. Relative risk of fever was 5.51 (95% confidence interval = 3.49–8.71) for AS01/AS02, 1.51 (0.46–4.90) for AS03, and 1.41 (0.83–2.40) for MF59 [80▪▪].

A similar study evaluating safety data of novel adjuvants among children 10 years of age or less demonstrated that local pain was the most frequent adverse event, with rates reported as 8.0–91.4% for AS01/AS02, 31.7–84.6% for AS03, and 1.0–59.0% for MF59, respectively [81]. The rate of Grade 3 pain, defined as spontaneous pain or which induce crying when children move their limb, reported in phase 3 trials was 0.1–0.3% for AS01, less than 1% for MF59, and 4.3–12.4% for AS03-adjuvanted vaccines, respectively [82–85].

The incidence of Grade 3 fever defined as more than 39 °C was 2.5% for AS01 among children 5–17 months of age and less than 1% among infants 6–12 weeks of age (consistent with the generally lower reactogenicity of vaccines in early life [86]), 1.9–5.4% for AS03, and 1% or less for MF59-adjuvanted vaccines, respectively [82–85,87,88].

While reactogenicity is defined as acute inflammatory reactions after vaccination, the term ‘safety’ refers to all adverse events attributable to vaccination that could potentially be caused, triggered, or worsened after vaccine administration [77▪▪]. In addition to the symptoms described above as reactogenicity, safety would include adverse events such as anaphylactic reactions after administration of an adjuvanted vaccine.

Although not proven via epidemiological research, and partially influenced by the immunomodulatory MOA of most adjuvants, concerns have been raised regarding the hypothetical potential of adjuvanted vaccine to increase autoimmune diseases [89–91]. Unexpected rare adverse events may also occur which are challenging to ascribe causality to.

For example, an increased risk of narcolepsy was observed in several European countries after AS03-adjuvanted 2009 H1N1 influenza vaccine, and alum has been linked to macrophagic myofasciitis, although underlying mechanisms and causality are not fully elucidated [91–96].

Because the sample size of prelicensure clinical trials is not usually sufficient to detect rare adverse events, it is essential to monitor postlicensure data (i.e., phase 4) with well selected controls [79▪]. Reporting potential vaccine side-effects, including attributable adverse events, is crucial to the integrity of vaccine development and will inform development of vaccines optimized safer vaccines via transparency and public awareness of possible vaccine-associated adverse events [97].

If an adverse event is associated with a given vaccine, it is important to assess which of the vaccine components, potentially including the adjuvant, may contribute to that adverse event. The Vaccines Adverse Events Reporting Systems (www.vaers.hhs.gov) is important for reporting, assessing and addressing vaccine safety.

WHO provides an e-learning course which is to establish a shared understanding among professionals whose work is linked to vaccine safety issues [98]. Prospectively designed vaccine studies in which systems biology assays are conducted on samples from rare events as compared with unaffected controls may represent an approach to get further insight into rare events.

MECHANISM OF REACTOGENICITY
After injection, adjuvants along with antigens rapidly stimulate the immune system. Adjuvants that are PRR agonists (e.g., MPLA, a TLR4A) directly activate innate immune cells, such as dendritic cells, and induce production of proinflammatory mediators including cytokines (e.g., IL-1β, IL-6, and tumor necrosis factor), chemokines (e.g., CCL2, CXCL1, and CXCL9), lipid mediators [e.g., prostaglandin-E2 (PGE2)], complement cascade components (e.g., C3a and C5a) and vasodilators (e.g., vasoactive amines and bradykinin) [77▪▪,99].

These soluble factors may sensitize peripheral nociceptive responses [100]. Adjuvants may drive injection site cell death/cytotoxicity and induce release of damage-associated molecular patterns (DAMPs) from injured/dead cells and tissue. DAMPs, such as chromatin-associated-protein-high-mobility group box 1, heat shock proteins, purine metabolites, and host cell DNA, can act as autologously derived endogenous adjuvants. Endogenous adjuvants may act in an additive or synergistic way with exogenous adjuvants [58,101].

Neutrophils, monocytes and lymphocytes accumulate at the site of immunization as early as 3–6 h after vaccine injection [26,99]. Vasodilators and chemokine promote cell recruitment from blood, but may also lead to the development of redness (erythema) and swelling. Most cytokines and chemokines in the injection site decrease within 24 h and reach baseline after 72 h [99,102,103].

For example, MF59 increases recruitment of immune cells into the injection site through secretion of chemokines, such as CCL2, CCL3, CCL4, and CXCL8 [76▪]. MF59 also accelerates and enhances monocyte differentiation into mature dendritic cells, and facilitates migration of dendritic cells into tissue-draining lymph nodes to prime adaptive immune responses [76▪].

Because each adjuvant has a distinct MOA that may further vary with vaccine dose and demographics of the target populations, including age [58,104], their contribution to reactogenicity symptoms may vary. Although these phenomena may be crucial for strong immunogenicity, these same inflammatory events may also lead to the development of local reactogenicity.

Systemic reactogenicity may be initiated when pyrogenic factors such as proinflammatory cytokines (e.g., IL-1β) and chemokines, and PGE2 are produced in sufficient levels to enter the systemic circulation and the central nervous system (Fig. 2). Within the brain, the coupled induction of the inducible enzymes cyclooxygenase-2 and microsomal PGE synthase-1 enhances intracerebral PGE2 concentrations, thereby causing an elevation of body temperature [105,106].

FIGURE 2
Medicinal chemistry and formulation can limit adjuvant-related systemic reactogenicity. (a) Free small molecule adjuvants are rapidly released and dissipated from the injection site. Entry of such adjuvants into the systemic circulation induces production of cytokines and prostaglandin-E2 thereby increasing systemic reactogenicity, including fever and malaise. (b) Lipidation of small molecules adjuvants, is one approach to reduce the systemic reactogenicity without reducing immunogenicity through the slow release of the adjuvant with low systemic distribution. PGE2, prostaglandin-E2. This figure was created using BioRender ( https://biorender.com/).

reference link : doi: 10.1097/MOP.0000000000000868


More information: Cameron C. Hanna el al., “Synthetic protein conjugate vaccines provide protection against Mycobacterium tuberculosis in mice,” PNAS (2020). www.pnas.org/cgi/doi/10.1073/pnas.2013730118

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