A simple addition to injected COVID-19 vaccines could enhance their effectiveness and provide “border protection” immunity in areas like the nose and mouth to supplement antibodies in the bloodstream, new research suggests.
The strategy involves dampening the activity of an enzyme produced by some white blood cells when they’re responding to the vaccine challenge. When highly active, this enzyme breaks down not just the pathogen – its job – but also degrades pieces of cells that participate in the immune response.
Research in mice showed that an experimental COVID-19 vaccine containing a compound to inhibit the enzyme stimulated a robust antibody response that included immunity in the nose and mouth, ultimately providing extra protection for airways and the gastrointestinal tract.
“Our approach is to improve ‘border control.’ The benefits are broad because in addition to providing protection in the bloodstream like most vaccines do, we also have excellent protection in the doors and windows of the body that communicate with the outside,” said senior study author Prosper Boyaka, professor and chair of the Department of Veterinary Biosciences at The Ohio State University.
“If we protect the mucosal area where the pathogen enters, then even if you don’t reach total immunity there, you limit the amount of pathogen that enters the body so the antibodies inside are more efficient at clearing the infection.”
The experimental vaccine was produced by packaging a segment of the SARS-CoV-2 (the virus that causes COVID-19) spike protein as an antigen with the common vaccine ingredient aluminum salts and an enzyme inhibitor.
The findings suggest this affordable design could be particularly helpful in developing countries, where cold storage needed for existing vaccines is a challenge, said Boyaka, also an investigator and program director in Ohio State’s Infectious Diseases Institute.
The study was published online Aug. 5 in Proceedings of the National Academy of Sciences.
There is an irony to the use of aluminum salts (also known as alum) in about 70% of the world’s vaccines: While alum’s presence actually enhances the immune response, it also recruits the white blood cells that secrete the enzyme, called elastase.
Alum is inexpensive to obtain or produce and can be stored at room temperature, and is effective at promoting development of a bloodstream-based antibody response to vaccination. But it doesn’t do much for cell-mediated immunity that improves protection against viruses and bacteria that use cells to reproduce, and can’t generate a useful number of antibodies in the body’s portals of entry for most pathogens: the nose, mouth and genitourinary tract.
The researchers found that suppressing elastase in a vaccine containing alum had the dual benefits of broadening and speeding up the antibody response in the bloodstream and triggering the specific types of antibodies needed for immune protection of mucous membranes.
“We found a way to have the cells come and help the immune response to develop and the enzyme to break down the pathogen, but we don’t want that response to be so high that it goes out of control. So we’re just putting a brake on the activity those enzymes would have,” Boyaka said. “And we found if you apply that strategy, you can induce a response in the airways even if the vaccine is not given through the airway.”
The experimental vaccine enhanced the magnitude of mouse antibodies, which reacted to the same section of the spike protein in the vaccine that antibodies in plasma from COVID-19 patients attach to, as well as generating antibodies in mucosal areas. Immunized mice lacking the gene for the enzyme developed high-affinity antibodies as well.
To further test the concept, the researchers found the enzyme-suppressing compound used in the study triggered production of specialized inflammation-regulating cells in cultures of human immune cells and pig spleen cells, showing that this strategy could improve vaccine immune responses in other species – including people.
“COVID will stay with us for some time, unfortunately, with the new variants,” he said. “What we need to do is have a portfolio of options that we could use depending on the health environment.
“Reprogramming the immune response induced by an injected vaccine containing alum is a way to make the vaccine more efficient for what we need. This could be a cheap and simple approach that can benefit people in developing countries.”
Coronaviruses (CoVs) are single-stranded RNA viruses characterized by club-like spikes that can potentially cause severe respiratory disease in humans (1, 2). The outbreak of severe acute respiratory syndrome (SARS) caused by the SARS-CoV resulted in more than 8000 confirmed infections, with an overall case fatality rate of 10% in 2002 (3). The Middle East respiratory syndrome (MERS)-CoV continues to cause deaths with increasing geographical distribution and a 34.4% case fatality rate, according to the World Health Organization (WHO) (4). Most recently, the coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has spread globally, with over 33 million confirmed cases as of October 2020 (5). Considering the challenges to global health systems and the far-reaching consequences on the world economy, there is an urgent need to develop effective and safe vaccines that can be quickly deployed on a global scale (2, 6).
Vaccine candidates are currently under development using different platforms, such as inactivated vaccines, recombinant protein vaccines, live-attenuated vaccines, viral vector (adenovirus) vaccines, DNA vaccines, and mRNA vaccines (2, 6, 7). Adenovirus-vector could induce potent immunological responses due to the presence of viral proteins and stimulation of innate immunity sensors, e.g., toll-like receptors (8).
Nucleic-acid vaccines, e.g., DNA and mRNA vaccines, encode the virus’s spike protein, intrinsically could engage innate immunity that instructs induction of immune protection (9). However, these platforms haven’t been used in licensed human vaccines before. In other platforms, subunit or inactivated antigens were used, but these antigens lack the immunological profiles that mediate the enhanced adaptive immunity. Thus, in these CoV vaccines, they require the addition of adjuvants for directing the types and magnitude of immune responses (10). In previously reported exploratory and pre-clinical CoV vaccine studies, adjuvants such as aluminum salts, emulsions, and toll-like receptor (TLR) agonists, have been used in vaccine formulations for studies with various animal models (Table 1).
The adjuvants AS03, MF59, and CpG 1018 have already been used in licensed vaccines (28) and have been committed by GlaxoSmithKline, Seqirus, and Dynavax to be available for COVID-19 vaccine development (29). When combined with subunit and specific inactivated antigens (30, 31), adjuvants with various characteristics elicit distinctive immunological profiles with regard to the direction, duration, and strength of immune responses. Thus far, there are at least 40 candidate vaccines in clinical trials and 149 vaccines in preclinical evaluation, of which 67 subunit and 15 inactivated COVID-19 vaccines have being developed (32).
Among these adjuvants, alum have been formulated with S protein or RBD to induce neutralizing antibody production (17, 18), which has suggested to be associated with protection against SARS-CoV-2 (15, 16, 24). However, alum lacks the capability to promote the activation of CD4+ and CD8+ T cell responses, which has been demonstrated to coordinate with the antibody responses to provide protective immunity against the SARS-CoV-2 (33).
Other adjuvants, e.g., emulsion adjuvants and TLR agonists, which have been shown to induce both humoral and cellular immune responses could be more favorable. However, no phase III clinical trial results of COVID-19 vaccines are published so far, thus, there is no direct evidence to indicate which type of immune response induced by vaccine plays a more critical protective role in SARS-CoV-2 infection. Knowing these uncertainties, an overview of previous CoV vaccine studies using different adjuvants would be indispensable for the design and development of a COVID-19 vaccine.
The SARS-CoV-2 is a novel strain of the coronavirus, and very little is known about its epidemiology and pathogenesis. Therefore, extreme cautions should be taken when considering vaccine formulations that can achieve the desired efficacy and safety profiles. The selection of adjuvants should consider the magnitude, affinity, isotype, and durability of antibodies that are critical for coronavirus vaccine developments (34).
It should be noted that low antibody production may lead to antibody-dependent enhancement (ADE) manifested by severe liver damage and enhanced infection (35), while high affinity neutralizing antibodies could help to avoid ADE. Additionally, the proper application of adjuvants also depends on the choice of antigens. The full-length S protein is more likely to trigger ADE due to mild antibody production (36). In comparison, the N protein is generally highly conserved, and it is associated with the ability to induce cytotoxic T lymphocytes (CTL). However, N protein could potentiate pro-inflammatory cytokine production and lead to severe lung pathology (37).
In addition, previous study on respiratory syncytial virus (RSV) vaccine also indicated that immunization with whole inactivated virus could lead to vaccine-associated enhanced respiratory disease (VAERD), manifested by allergic inflammation and Th2 type immune responses (38). Altogether, these studies suggest that vaccines formulated with various antigen isotypes may require proper adjuvant selection to achieve the desired immune protection. In this paper, we reviewed adjuvants that have already been incorporated in the coronavirus vaccines under exploratory and pre-clinical investigations. By reviewing the vaccine formulations and the types of immune responses that were induced, we provide information that will enable proper adjuvant selection for COVID-19 vaccines to facilitate rapid vaccine delivery.
Aluminum Salt-Based Adjuvants
Aluminum salt-based adjuvants (alum) were the first adjuvants used in licensed human vaccines. They are still the most widely used because of their wide-spectrum ability to strengthen immune responses and their excellent track record of safety (39–41). In limited coronavirus vaccine studies, it has been suggested that neutralizing antibody against the spike protein might be mechanistically correlated with immune protection (42). When alum was formulated with S protein or receptor-binding domain (RBD), it significantly enhanced humoral immune responses.
This was demonstrated by higher titers of serum IgG1, increased high affinity viral neutralizing antibodies, and the generation of long-lasting memory B cells in mice (13, 17–19). Additionally, Alum was formulated with the inactivated and VLP vaccines containing E, M, and N proteins (11, 12, 14) (Table 1) that showed enhanced IgG1 and neutralizing antibody titers (14) and prolonged durability (12). Studies also demonstrated that alum adjuvant plays an essential role in the dose-sparing of CoV vaccines. In a SARS S protein subunit vaccine, the alum-adjuvanted S protein (1 μg) group showed neutralizing antibody titers similar to or higher than the non-adjuvanted S protein (50 μg) group.
The alum-adjuvanted S protein (5 μg) group showed a geometric mean titer (GMT) twice as high as the non-adjuvanted S protein (50 μg) group (20). It should also be noted that different types of alum were selected in the studies, including Alhydrogel, which is chemically crystalline aluminum oxyhydroxide (43), aluminum hydroxide (11), aluminum phosphate (22), and Imject™ Alum (23), which is a mixture of aluminum hydroxide and magnesium hydroxide. Even though there is no specific description regarding the aluminum hydroxide in reported literature (11, 18, 21), it can also be referred to Aluminum oxyhydroxide (44). However, these studies lacked systematic comparisons with regards to their adjuvanticity and how various alum-based adjuvants differed in their ability to induce neutralizing antibodies.
It is worth noting that inactivated SARS-CoV or S protein-based vaccines are associated with Th2-type immunopathology, which is characterized by an increase in eosinophils and inflammatory infiltrates (14, 30, 37, 45). Moreover, the addition of alum adjuvant exacerbated the immunopathologic reactions (14, 45). In alum-adjuvanted SARS-CoV double-inactivated vaccine (DIV), there was a skew in the N or S protein-specific antibodies toward IgG1, when compared with the more balanced antibody production in the nonadjuvanted DIV vaccine (14).
These observations raise significant concerns regarding the safety of adjuvanted coronavirus vaccines. On the other hand, it has been shown that alum can reduce immunopathology in SARS-CoV vaccines containing either a double-inactivated virus or S protein (11). Furthermore, in a recent study, a purified inactivated SARS-CoV-2 vaccine (PiCoVacc) adjuvanted with aluminum hydroxide conferred complete protection in non-human primates (rhesus macaques) with potent humoral responses but without lung immunopathology (15).
This finding raises the question of the mechanism of eosinophilic immunopathology. While commonly thought of as the product of Th2 responses, recent studies have indicated that tissue eosinophilia can also be controlled by Th17 responses (46). Thus, the proper selection of CoV antigens and adjuvants that can shift host responses away from a Th17-bias appears to be critical. In addition, other studies have demonstrated that the Th2 immunopathology may be associated with SARS N or S protein that results in enhanced eosinophilic immunopathology (11, 37, 47). However, more studies are required, as the preliminary data is limited. Additionally, the Th-2–biased immune responses may raise the concern on vaccine-enhanced respiratory disease (VAERD) (38, 48), however, there are no evidences that alum-adjuvanted CoV vaccines show the effect.
When alum was used as an adjuvant in CoV vaccines (Table 1), there was a lack of Th1 CD4+ T cell and cytotoxic CD8+ T cell immune responses, which is typical for alum-adjuvanted vaccines (49). However, recent study has demonstrated that the SARS-CoV-2–specific adaptive immune response correlated with milder disease, indicating that coordinated CD4+ and CD8+ T cell responses play a synergistic effect in the protective immunity of COVID-19 (33). Several other adjuvants, which are capable of inducing more balanced Th1/Th2 or Th1-biased immune responses, have been formulated in CoV vaccines and will be discussed in the following sections.
The emulsion adjuvants, MF59, and AS03 have already been used in licensed human vaccines to improve the immunogenicity of the antigens (50, 51). Compared with alum that lacks the capability to mediate cell-mediated immunity (49), MF59 and AS03 can elicit more balanced immunity, possibly by improving antigen uptake, recruiting immune cells, and promoting the migration of activated antigen-presenting cells (28, 50, 52). Emulsion adjuvants have already been used in preclinical studies of vaccines against coronavirus. MF59 used in inactivated SARS and MERS vaccines, as well as vaccines containing the RBD domain of the MERS-CoV spike (S) protein, has exhibited excellent adjuvanticity, with potent humoral immune responses, i.e., high titers of neutralizing antibodies, and cell-mediated immunity in the coronavirus vaccines (53–55).
In addition, depending on the types of antigen, cell-mediated immunity induced by MF59 differs. When formulated with the MERS-CoV S protein, MF59 enhanced both effective CD4+ and CD8+ T-cell immune responses. In comparison, when combined with inactivated SARS CoV, MF59 induced significant CD4+ T cell, but not CD8+ T cell responses (56, 57). However, in another study by Zhang et al., it was demonstrated that when MERS S protein was adjuvanted with MF59, it induced higher IgG1 and IgG2a antibodies with a slightly Th2-biased response (54).
Subsequent studies also showed that ferritin-based MERS-CoV S protein, adjuvanted with MF59, promoted multiple antibody responses, including high levels of IgA antibody titers that resulted in potent mucosal immune responses (58). A study by Tang et al. has indicated that there are no significant differences in the neutralizing activity of the serum derived from mice immunized with MERS S377-588 at 1, 5, and 20 μg in the presence of MF59, suggesting the dose-sparing effect of MF59 when it was formulated with MERS S protein (57).
However, an immunopathologic lung reaction, as well as an increase in IL-5 and IL-13 cytokines, was seen in animal studies using both MF59-adjuvanted and adjuvant-free inactivated MERS-CoV vaccines (53). It has shown that eosinophil infiltrations with higher Th2-type cytokine secretion aggravated the hypersensitivity-type pulmonary immunopathology when vaccinated with MF59-adjuvanted inactivated virus vaccines as compared with the inactivated virus vaccines alone (53).
Another emulsion adjuvant, AS03, elicits both potent humoral and cellular immune responses to an inactivated whole virion SARS-CoV (WI-SARS) vaccines (59) compared with the virion without adjuvants. Moreover, in the presence of the AS03 adjuvant, an identical trend toward specific CD4+ T cell responses was observed when immunized with SARS-CoV containing the equivalent of 0.5 or 1.5 μg of S protein (59). Therefore, the addition of AS03 tends to potentiate the immune responses with a lower dosage of antigen. Considering its capability to induce both arms of the immune system, S protein, RBD domain, and N protein can also be formulated with AS03. Currently, GSK is sharing its AS03 adjuvant with COVID-19 vaccine developers globally (29).
Besides MF59 and AS03, other emulsion-based adjuvants such as Freund’s adjuvant and Montanide ISA51 have also been formulated in CoV vaccines (54). By evaluating the titers of specific serum antibody responses, it has been demonstrated that Freund’s adjuvant and ISA51 elicited significant Th1 antibody responses (IgG2a) with no clear Th2 responses (IgG1) (54, 59).
TLR Agonists and Other Adjuvants
Toll-like receptors (TLRs), a category of pattern-recognition receptors, are critical to pathogen recognition. This allows for rapid activation of innate immunity, and subsequently, effective adaptive immunity. TLR agonists have been extensively studied as vaccine adjuvants (60, 61). CpG, Poly I:C, glucopyranosyl lipid A (GLA), and resiquimod (R848) are agonists for TLR9, TLR3, TLR4, and TLR7/8, respectively. These adjuvants have been evaluated in candidate vaccines against SARS CoV (62, 63).
In addition to neutralizing antibodies and CD4+ T cells, optimal protection against coronavirus probably involves the synergistic effect of CD8+ T cells (64). Memory CD8+ T cells solve the problem of neutralizing antibodies only existing for short periods and providing long-term protective cellular immunity (64). Among the TLR agonists, CpG significantly augments the CD8+ T cell immune response higher than the others (63). Indeed, it has been demonstrated that CpG can also stimulate enhanced IgG production in animals immunized with an inactivated SARS-CoV vaccine (62).
In addition to IgG, IgA production was also enhanced, only when CpG was administered via intranasal (i.n.) administration (62), indicating immune activation in the mucosal compartment. Although CpG is capable of inducing both cellular and humoral immune responses, it preferentially induces responses that are Th1-biased. Moreover, CpG can divert pre-existing Th2 responses to a Th1 phenotype, which has laid a foundation for the combination of CpG with other adjuvants, most commonly alum (65). In SARS-CoV or MERS-CoV subunit vaccines, studies have found that the combination of alum and CpG elicited higher neutralization antibody titers and a more robust cellular immune response compared with alum alone or alum with other TLR agonists (18, 19).
In addition to alum, CpG is combined with Montanide ISA-51, a type of water-in-oil emulsion adjuvant. When the combined adjuvants were formulated with SARS S or N protein, they were capable of promoting robust neutralizing antibody production (66). However, vaccinated with only SARS N protein, animals showed immune responses biased dramatically toward Th1 (67). In addition, it is reported that R848 could enhance antigen-specific CTL response and induce a fast, robust and durable IFN-α production in vivo among humanized mice, which is distinct from the experimental findings based on common mouse models (68).
However, further studies on R848 adjuvanticity should stress more on vaccine formulation. A recent study by Gadd et al. indicated that only when R848 was conjugated with DOPE (1,2−di−(9Z−octadecenoyl)−sn−glycero−3−phosphoethanolamine):DDA (dimethyldioctadecylammonium bromide salt) multilamella liposomes rather than linear mixed, a high potency of immunostimulatory activity was observed (69). Moreover, an R848-encapsulating PLGA nanoparticle can bring down the excessive level of inflammatory cytokines induced by free R848, which could be benefit to provide long-term safety and appropriate immune response (70).
Although CpG had been shown to exhibit considerable potential as a coronavirus-specific adjuvant, studies have found that it might be a poor inducer of long-term immune memory (46). A recent study indicated that single-stranded RNAs (ssRNAs) derived from the Cricket paralysis virus (CrPV) intergenic region (IGR) internal ribosome entry sites (IRES) could function as vaccine adjuvants endowing long-lasting immunity. This adjuvant significantly activates innate immune response through activating TLR7 and enhancing the chemotaxis of professional antigen-presenting cells (APC) (71).
Moreover, some novel adjuvants such as STING agonist, Advax, and AS01B, which is an adjuvant formulated in recombinant zoster vaccine Shingrix, exhibit advantages for long-lasting immune responses (46, 59, 72). Advax, a delta inulin microparticle adjuvant, augmented the induction of neutralizing antibodies along with the existence of memory B cells and a robust, long-lasting T-cell IFN-γ response when it was formulated in recombinant or inactivated SARS-CoV vaccines (46). Moreover, Matrix M1, a saponin-based adjuvant, has been demonstrated to be more effective than alum adjuvant in inducing neutralizing antibodies to SARS S protein or MERS S protein (17). This might address the concern that S protein may lead to antibody-dependent enhancement (ADE), which is more likely to be triggered by mild antibody production (36).
The SARS-CoV-2 infections occur at the mucosal surface of the upper respiratory tract (73). Thus, the elicitation of protective immune responses at the mucosa is critical. TLR agonists, such as flagellin (74) and CpG ODN (62), have been used as mucosal adjuvants. As discussed above, the CpG ODN can elicit neutralizing antibodies in mucosal compartments (62) when formulated with inactivated SARS-CoV. Additionally, the STING agonist, bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP or cdGMP), has been reported as a potent mucosal vaccine adjuvant that induces Th1 and Th17 cytokines in a plant-derived H5 influenza vaccine after intranasal vaccination (75).
In a very recent study, it was demonstrated that pulmonary surfactant–biomimetic liposomes encapsulating STING agonists could be used as mucosal adjuvants for universal influenza vaccines that trigger rapid humoral and cellular immune responses and exhibit sustained cross-protection against influenza (76). Though cdGMP in polymeric nanoparticle formulations has been used as adjuvants with MERS-CoV S-RBD protein, its ability to induce mucosal immunity was not specifically examined (72). Thus, further studies are warranted to examine both the efficacy and safety of mucosal adjuvants in coronavirus vaccines.
reference link: https://www.frontiersin.org/articles/10.3389/fimmu.2020.589833/full
More information: Eunsoo Kim et al, Inhibition of elastase enhances the adjuvanticity of alum and promotes anti–SARS-CoV-2 systemic and mucosal immunity, Proceedings of the National Academy of Sciences (2021). DOI: 10.1073/pnas.2102435118