Opinion: Making Inactivated and Subunit-Based Vaccines Work

Abstract

Empirically derived vaccines have in the past relied on the isolation and growth of disease-causing microorganisms that are then inactivated or attenuated before being administered. This is often done without prior knowledge of the mechanisms involved in conferring protective immunity. Recent advances in scientific technologies and in our knowledge of how protective immune responses are induced enable us to rationally design novel and safer vaccination strategies. Such advances have accelerated the development of inactivated whole-organism- and subunit-based vaccines. In this review, we discuss ideal attributes and criteria that need to be considered for the development of vaccines and some existing vaccine platforms. We focus on inactivated vaccines against influenza virus and ways by which vaccine efficacy can be improved with the use of adjuvants and Toll-like receptor-2 signaling.

Introduction

A part from the availability of clean water and the implementation of good hygiene practices, when implemented by organized programs, vaccination is the most successful and economically effective strategy to significantly reduce the mortality and morbidity caused by infectious diseases (60,87,89). The idea that immunization can lead to protection against a future encounter with a pathogen was first recognized 1,000 years ago through the practice of variolation with live smallpox virus in China and subsequently through the pioneering work of Edward Jenner in 1798, who was the first to confer scientific status and principles on the procedure. Since then, the development of vaccines has grown rapidly especially in the late 19th and 20th centuries paralleling major discoveries in microbiology and immunology.

Vaccines developed before the 1980s used largely empirical approaches relying on the use of disease-causing microorganisms that were inactivated (usually by heat or chemical treatment) or attenuated (generally by passaging through animals, cell lines, or exposure to unfavorable growth conditions) before being injected and often without prior knowledge of the mechanisms involved in conferring immunity (29). The use of live- and attenuated- or killed whole-organism-based vaccines had enormous successes in the control and even eradication of a number of common and sometimes lethal human diseases, including smallpox (14), polio (63), measles (10,79), mumps (57), and rubella (103).

Vaccination using live attenuated vaccines established an effective way to elicit the breadth and magnitude of immune responses that may mimic the state of protective immunity that follows natural infection. However, concerns surrounding live attenuated vaccine use include (i) the potential to cause disease in immunocompromised individuals and (ii) the possibility of reversion to a virulent form as a result of back-mutation of attenuating mutations, acquisition of compensatory mutations, and/or recombination with circulating transmissible wild-type strains. Although these concerns have led to development of new ways to rationally attenuate an organism to minimize any tendency for reversion without compromising efficacy, they have also prompted and accelerated the development of inactivated whole-organism- and subunit-based approaches. The down side of course is that although there is little or no chance of reversion to the wild-type organism, the ability to elicit a state of immunity, particularly in the case of subunit-based vaccines, is often low.

In the case of vaccines that depend on the induction of neutralizing antibodies for protective effect, antibodies elicited by vaccines may have low to nonexistent efficacy in the context of antigenically hypervariable pathogens such as influenza virus (122), human immunodeficiency virus (HIV) (118), and malaria (16) and even vaccines that elicit CD8⁺ T cell responses are not immune from selection of vaccine escape mutants (4,91,). Vaccines that are designed to prevent diseases caused by pathogens capable of altering their antigenic signatures therefore require continuing vigilance of the pathogen landscape, as is the case with influenza surveillance programs, and/or the discovery of ways in which to elicit antibodies and/or cell-mediated immunity against nonvariable antigens (i.e., conserved regions, and structurally or functionally essential regions of the target antigens).

The poor immunogenicity of many subunit-based vaccines may demand that large doses and/or inclusion of an adjuvant are necessary to accelerate and amplify the ensuing immune response. These requirements also introduce problems because many adjuvants are toxic and only a very few of those that are licensed for use in humans demonstrate efficacy and safety (1).

As the pharmaceutical industry moves toward defined immunogens and adjuvants to accommodate increasingly stringent regulations for vaccines, so has the immunogenicity of vaccine decreased as the “danger signals” associated with the whole organism are removed during the purification processes. We have, however, made advances in immunology and have increased our understanding of how the innate immune system influences adaptive immunity. Using this knowledge, we now have insights into how to stimulate particular immunological pathways. The development of new adjuvants, determination of associated mechanisms of action, and improvements in antigen characterization techniques are leading us away from empirical design of vaccines toward the use of rational and tailored approaches. New approaches can result in the induction of efficacious immune responses without toxic or unwanted side effects.

Requirements of an Ideal Vaccine

The following is a list of preferable attributes for, but not limited to, inactivated or subunit-based vaccines. While it may not be likely that all conditions will be met by all vaccines, at the very least safety and efficacy are essential:

(i) The vaccine must be safe and be minimally toxic, producing few or no site reactions. Clearly, vaccination should not cause painful or unsightly site reactions, but the fact is that an inflammatory response is often or always the hallmark of initiation of an immune response and it is almost inevitable that some site reactions will result following vaccination. Of more importance are the toxic effects that may accompany vaccination. These can often be attributed to incomplete inactivation of the pathogen in the case of pathogen-derived vaccines (85) or the induction of inappropriate off-target or autoimmune responses (see also ii below).

(ii) The vaccine must be of well-defined specificity, that is, it must not elicit deleterious effects such as autoimmunity. Protein antigens obtained from pathogens can contain sequences that are sufficiently similar to the host proteins that antibodies elicited by the vaccine may cross-react with host proteins. For example, a major target for vaccines against Group A streptococcal infections is the M protein (26) and an attractive target sequence within this protein is the conserved C-terminal portion with sequence LRRDLDASREAKKQVEKALE. This sequence, however, contains epitopes that may elicit T cells cross-reactive with myosin of cardiac muscle (92). Obviously, the risk of inducing such an autoimmune reaction must be avoided, which requires that thorough engineering of this sequence removes any possibility of inducing cardiac muscle reactive antibodies, or a search for alternative targets (24,27,35,54).

(iii) The vaccine must be efficacious in the target species. Many vaccine studies are carried out in animal models that are inappropriate for the disease under investigation. For example, HIV, malaria, and influenza are not natural diseases of mice, but vaccine studies are usually done in these animals (3,50,80). The use of primates, including humans, may be prohibitive in preliminary investigations of vaccines for use in man, but at some point, evaluation in the target species will be necessary for candidate vaccines.

(iv) The vaccine should induce protective immunity, whether antibody based and/or cell mediated. Vaccine candidates traditionally elicit antibodies that neutralize the pathogen against which they are directed. Examples include vaccines against polio, smallpox, influenza, diphtheria, and tetanus. The idea is that antibodies will prevent disease by processes, including opsonization, prevention of virus binding to and infection of susceptible host cells, and neutralization of toxins produced by invading microorganisms. In these cases, it is clear that antibodies are sufficient and appropriate for protection. In many other cases, however, cell-mediated immune responses are necessary for protection. For viral diseases and cancer, it is often necessary to elicit cytotoxic T cells that are able to recognize the virus-infected or cancer cells to limit disease.

(v) The vaccine must be simple, economical, and lend itself to industrial manufacturing processes. Many ingenious but very complex vaccine candidates are described in the scientific literature but to attract manufacturers, vaccine preparation must be straightforward and economical. Furthermore, the governments and regulatory agencies that sanction and support vaccine manufacture and distribution must be convinced of vaccine worth to the community, and they must demonstrate how the vaccine benefits the economy (e.g., by sustaining a healthy workforce). This means that vaccines should be simple to make, lending themselves to the high-volume processes of modern-day pharmaceutical companies.

(vi) The vaccine should induce durable immune responses. Not only must vaccines provide rapid protection by mounting a speedy immune response but they must also provide lasting immunity. This is often achieved through use of multiple doses of vaccine being administered to bring about continuity of immunity as in the case of childhood vaccines and even adult vaccines, which require multiple visits to the clinic for repeat doses to maintain efficacy (see also vii below).

(vii) The vaccine should require minimal dosing to support vaccination compliance. The compliance required for immunity is often difficult to realize particularly in remote areas where repeated visits to a clinic are not feasible. Most vaccines require multiple doses for efficacy and the problem of compliance would be well served if a single dose of vaccine could provide many years of immunity.

(viii) The vaccine must have a clearly understood mechanism of action. The rigorous requirements of regulatory authorities, which require disclosure of mechanisms of action, are a significant hurdle to the marketing of vaccines. The demand for thorough and forensically detailed documentation describing new drugs and biologicals imposes an extra level of thoroughness in the scientific method that must be followed to bring new materials to market. This level of thoroughness was not previously necessary with many empirically derived vaccines.

(ix) Vaccine delivery platforms should be applicable to multiple indications. For example, new adjuvants should lend themselves to use in a variety of different vaccines, including vaccines that target bacteria, viruses, cancer, or normal physiological processes such as reproduction (127,128).

Current Vaccine Technologies

Over recent decades, our understanding of the immune system, including the different states of immunity that can be elicited and the development of new laboratory and industrial techniques in microbiology and immunology, has grown and as a consequence, vaccine design can now make use of nonempirical approaches that use specific antigen targets together with safe and efficient modes of delivery using, for example, rationally designed adjuvants and other delivery systems.

Advances in manufacturing-scale recombinant protein-based technologies have enabled the production of many different whole and partial antigen-based vaccine candidates ranging from complex structures, including the virus-like particles (VLPs) of human papillomavirus (HPV) (49,129) and the subviral particles of hepatitis B virus (HBsAg) produced in yeast (74,111). Each of these vaccines is particle based and it should be remembered that by virtue of their size, some particles are themselves a danger signal to phagocytic cells of the innate immune system. At the other end of the size scale are peptide-based vaccines. Our knowledge of the amino acid sequences of individual epitopes of pathogens that are recognized by antibodies and/or T cells has enabled us to chemically synthesize epitope-based vaccines (8,34,59,126).

Complex mixtures of antigens, including inactivated influenza detergent-disrupted vaccines and the trivalent measles, mumps, and rubella vaccine, demonstrate our ability to induce immunity against a variety of different antigens simultaneously by a single inoculation. Of significance are glycoconjugate vaccines against Haemophilus influenzae type B and various meningococcus and pneumococcus serotypes comprising capsule polysaccharide antigens. These are derived from whole organisms and are then conjugated to diphtheria toxoid carriers to support T cell help. More recently, genomics-based antigen discovery and in silico predictive approaches have also been utilized to successfully identify protective antigenic determinants from meningococcus serotype B that would eventually lead to the licensing of an effective vaccine. These and other approaches now form an important part of many vaccine developmental strategies against microorganisms, including Mycobacterium tuberculosis, Porphyromonas gingivalis, group A streptococcus, as well as antibiotic-resistant bacterial strains.

There is no doubt that we know enough about the immune system to be able to translate this knowledge into some successful vaccines. There is, however, a great deal yet to do because as yet there are still many infectious diseases that we need protection against and for which there are no licensed vaccines [e.g., chikungunya (117) and hookworm (12)]. In addition, there is a fast approaching situation through which microorganisms are becoming resistant to multiple and even all antibiotics in the pharmaceutical pipeline. Protective strategies against noninfectious diseases, including cancer, for which there is increasing evidence that immunotherapies could be successful (61,97) are also waiting to be discovered. We do not yet know enough to address all the problems, but there are some very encouraging discoveries and technologies that are leading the way.

A Case Study—Influenza Vaccines

Despite the availability of a new vaccine every year, influenza continues to have a significant impact on global health causing 250,000–500,000 deaths and up to 5 million cases of severe illnesses annually (120). In the United States alone, the economic impact caused by seasonal influenza is estimated to cost $26.8–$87.1 billion a year (78) with the expectation that this figure will increase in the event of a pandemic outbreak.

The most commonly used vaccine against seasonal influenza is based on inactivated, detergent-disrupted, or split virions, which induce neutralizing antibodies against the viral surface glycoproteins, hemagglutinin and neuraminidase. These vaccines are usually available as trivalent or quadrivalent formulations (containing 2 influenza A strains in combination with 1 or 2 influenza B strains). Their effectiveness is largely dependent on how correctly matched the vaccine strains are to those that are predicted to circulate in the upcoming influenza season. As a result of continual viral antigenic drift and/or shift, the efficacies of these vaccines wane over time and need to be reformulated regularly to maintain protection of the target population.

While the CDC estimates for the 2015–2016 seasons indicate that influenza vaccination prevented ∼5.1 million illnesses, 2.5 million medically attended illnesses, and 71,000 hospitalizations associated with influenza (96), recent human outbreaks of the avian-derived strains H7N9 in 2013 (43), H5N1 in 2004 (67,114) and the swine-derived H1N1 pandemic strain in 2009 (18) also highlight deficiencies in our ability to protect against the emergence of novel influenza viral subtypes.

A significant impediment to our ability to respond to a potential influenza outbreak sufficiently and quickly is the time that it takes for a vaccine to be developed and manufactured. Production of sufficient amounts of purified virus to make a vaccine is reliant on virus growth in embryonated eggs, which can take some 6 months to reach sufficient manufacturing capacity (48). Advances in vaccine technologies and the establishment of new production facilities in developing countries have increased current global capacity to manufacture seasonal trivalent vaccines from 500 million doses in 2006 up to 1.5 billion doses in 2013 and it is estimated that 6.2 billion doses of monovalent pandemic vaccine, sufficient to cover the world's population, could be made (119).

Nevertheless, vaccine shortages are predicted to occur after the outbreak of a strain to which the population is immunologically naive. In individuals with little or no pre-existing immunity against a pandemic influenza virus, H5N1 or H7N9, for example, it is likely that two vaccine doses will be needed to elicit sufficient protection (7,9,25,109). It is also important to remember that resources used to produce a pandemic vaccine should not be at the detriment of the manufacture of vaccines against seasonal strains. Clearly, the development of improved vaccine technologies and more effective vaccine delivery systems that provide protection with smaller or single doses of vaccine would provide an enormous advantage in times of greatest need.

Another limiting factor of existing split-virus vaccines is the vaccine's inability to induce effective cell-mediated responses, especially CD8⁺ T cell responses. The potential of these responses, particularly if directed against internal viral proteins, including the nucleoprotein (NP) (52) and the matrix protein (M1) (51), which are relatively conserved across heterologous strains, has been shown to be associated with better prognosis following influenza outbreaks (37,75,100). In the case of zoonotic H7N9 infections in humans, Wang et al. have also found that the presence of an early cross-protective CD8⁺ T cell response plays a key role in mediating fast recovery from acute infections (116), further illustrating the importance of CD8⁺ T cell responses in resolving influenza infection.

In recent years, a variety of different forms of influenza vaccines have been investigated in attempts to improve their ability to protect against influenza, and a number of studies have revisited the use of whole inactivated influenza virus as a vaccine. The foundation for this follows reports that whole-virus-based vaccines are more immunogenic than split-virus or subunit-based vaccines, particularly in immunologically naive humans and animals (47,55,81,86,102). Importantly, the concerns of the high reactogenicity that has been reported to be associated with the use of whole-virus-based vaccines, thought to be the result of residual egg-derived products carried through from the production process (90), have now been lessened through the implementation of better purification techniques and/or the use of cell culture-based production systems (5,6,11). Several clinical trials have since reported a similar reactogenicity of whole-virus compared with split-virus-based vaccines (36,68,70,83,112,115) and some of these studies also reported higher seroconversion rates using lower doses of whole-virus compared with split-virus vaccines (36,68,70).

It has been suggested that the dose-sparing effects could be due to the RNA in whole-virus vaccines that signals through TLR7 and provides an adjuvant effect (17,47). It could also be that particulate whole virus possesses a higher density of antigenic proteins than detergent-solubilized antigens of a split-virus vaccine (46,47). In addition, the inactivation of whole-virus vaccines by γ-irradiation may preserve antigenic and immunogenic domains of viral proteins better than chemical inactivation techniques (e.g., formalin or β-propiolactone treatments used in split- and whole-virus vaccines) (42). Clearly, the dose-sparing potential and improved immunogenic properties offered by the whole-virus vaccines over the split-virus vaccines make whole-virus vaccines attractive candidates for further investigation. In addition, the production of whole-virus vaccines uses a relatively simple manufacturing process that is especially attractive when mass vaccination is required within a short period of time.

Adjuvants and Vaccine Delivery Systems

Although we may know what the important antigens, and consequently, the vaccine targets of a pathogen may be, many protein-based and epitope-based vaccines suffer from the disadvantage that they are usually unable to trigger an immune response without adjuvant. Currently, a very small number of adjuvants have been licensed for human use and among these, the aluminum-based adjuvant alum has been used for 80 years in vaccine formulations, especially in subunit-based vaccines, including diphtheria toxoid, tetanus toxoid, the acellular pertussis vaccine (Daptacel) and hepatitis B vaccine (Engerix-B) [reviewed in Ref. (64)]. Until recently, alum was the only adjuvant approved for human use in the United States despite the fact that its mechanism of action was incompletely understood (40,72). Organic adjuvants such as oil-in-water emulsions (MF59 and AS03) have been recently licensed for use with inactivated split influenza vaccines in Europe (Fluad) (45,113) and are licensed in the United States for use in vaccines against pandemic strains of influenza virus (Prepandrix) (19). AS04, a combination adjuvant of monophosphoryl lipid A and alum, is approved for use with the HPV vaccine (Cervarix) in Europe (33,88). The use of adjuvants in vaccines through stimulation of the immune response can improve vaccine efficacy, but because of their immunostimulatory nature they must be used with care, so that overproduction of proinflammatory molecules, which can lead to overt inflammatory reactions or induction of autoimmunity, is avoided (93,98).

The discovery of pattern recognition receptors (PRRs) has helped enormously in the rational design of vaccines, particularly the ways in which vaccines are adjuvanted. PRRs, which have evolved to recognize and respond to molecules and patterns of molecules present on many pathogens, include the family of Toll-like receptors (TLRs). Because the innate immune system determines the nature of adaptive immune responses, the agonists, molecules recognized by PRRs, have been subjects of great interest as potential adjuvants. They include di- and triacylated palmitic acid-based derivatives, which have been used to target TLR2 (21,58,59,124,127), the TLR3 agonist Poly-IC (71,101,110), Monophosphoryl lipid A for TLR4 (44), flagellins for TLR5 (82,106), imidazoquinolines for TLR7/8 (62), and CpG oligodeoxynucleotides for TLR9 (15,73). Saponins (32) and bacterial exotoxins, including heat-labile toxins, (84) have also been studied as potential adjuvants. Other experimental antigen delivery approaches include those that target C-type lectin-based receptors such as the mannose receptor using mannosylated ligands (2,94), dectin-1 with its agonist β-glucan (69,123), as well as Mincle using trehalose-6,6′-dimycolate (28). In addition, novel strategies using antibodies or ligands directed against DC-SIGN (39,104), Langerin (38), XCR-1 (41,53), and Clec9A (66,95) are also being used to pave the way for targeting specific dendritic cell populations to direct antigen into cross-presentation pathways.

Targeting Antigen Delivery to TLR2 with Pam₂Cys-Based Adjuvants

Of great interest and focus to us is the use of the TLR2 agonist S-[2,3-bis(palmitoyloxy)propyl]-cysteine or Pam₂Cys as a vaccine adjuvant (20 –22,59,65,99,121,124,125). Covalent attachment of Pam₂Cys to peptide epitopes can facilitate epitope delivery into dendritic cells, induce cell maturation, activate transcription factors such as NF-κβ, and cause secretion of proinflammatory cytokines (23,59,65). Lipidated peptide-based vaccines, which contain CD8⁺ T cell epitopes derived from influenza virus, Listeria monocytogenes and ovalbumin, but not their nonlipidated counterparts, are able to induce cell-mediated immunity that can protect against viral, bacterial, and tumorigenic challenge (30,31,59,65). Furthermore, vaccines containing B cell epitopes from hepatitis C virus (107,108), group A streptococcus (13), gastrin (59), or luteinizing hormone releasing hormone induce high antibody titers, which in the last example also results in abrogation of reproductive function (23,59,127).

Noncovalent association between Pam₂Cys and antigen has also been demonstrated and exploited as a promising strategy to induce immune responses without the complexities associated with covalent conjugation chemistries. We have demonstrated that the use of a branched cationic lipopeptide, R₄Pam₂Cys, which associates electrostatically with what would otherwise be nonimmunogenic protein antigens, enhances immunogenicity, inducing effective antibodies in addition to CD8⁺ T cell effector and memory recall responses (20 –22,99,121). The ability of this Pam₂Cys-based adjuvant to associate with antigen in a noncovalent manner circumvents the need for chemical modifications and associated processes that are often required for formulation of antigen and adjuvant, greatly simplifying vaccine manufacture.

An important advantage of our approach is that intranasal administration to animals of a low dose of inactivated detergent-split influenza vaccine formulated with R₄Pam₂Cys simultaneously stimulates innate and adaptive immune systems. Stimulation of the innate immune system by the intranasal route provides immediate antiviral protection, and antigen-specific immune responses that are stimulated then provide antibody- and cell-mediated immunity, both of which exhibit long-term memory. This particular influenza vaccine candidate provides rapid protection against challenge with homologous and serologically distinct influenza viral strains within a day of administration and lasts for up to a week (105) [thus acting as an antiviral, but administration also induces high levels of systemic and pulmonary-associated antibodies that protect against a vaccine-matched viral strain (22)]. In addition, improved primary virus NP-specific CD8⁺ T cell responses are induced providing heterologous protection against challenge with a nonmatched strain and reducing transmission to naive animals (22). Harnessing innate and adaptive immune responses with a single subunit-based vaccine formulation could be beneficial for providing community protection, particularly during periods between the onset of an outbreak and the time when a vaccine becomes available or in scenarios in which mass vaccination with a strain to which the population is immunologically naive is imperative.

Studies on the mechanisms that underpin innate immunity are providing a plethora of opportunities for rational design of vaccine delivery systems and adjuvants that may also provide immediate protective responses en route to induction of durable immunity mediated by antibodies and T cells. Our own studies using the TLR-2 agonist Pam₂Cys have indicated opportunities for T cell-based vaccines that induce cross-subtype protection against influenza A virus (22,56,76,77,105) and that provide immediate protection against influenza viruses through activation of a rapid and effective innate immune response. While our own work has focused on subunit-based vaccines against influenza, we propose that other respiratory pathogens can be stopped at their portal of entry using a similar approach.

Footnotes

Author Disclosure Statement

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in this article.

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Opinion: Making Inactivated and Subunit-Based Vaccines Work

Abstract

Introduction

Requirements of an Ideal Vaccine

Current Vaccine Technologies

A Case Study—Influenza Vaccines

Adjuvants and Vaccine Delivery Systems

Targeting Antigen Delivery to TLR2 with Pam2Cys-Based Adjuvants

Footnotes

Author Disclosure Statement

References

Targeting Antigen Delivery to TLR2 with Pam₂Cys-Based Adjuvants