The promise and challenge of epitope-focused vaccines

Abstract

Traditional vaccination with whole pathogens or pathogen-derived subunits has completely eliminated diseases like smallpox, and has greatly limited the incidence, morbidity and mortality associated with many other infectious diseases. Unfortunately, a large burden of infectious disease remains that may be preventable through vaccination. For many of these, more focused and innovative approaches may be essential for the development of effective vaccines.

Keywords:

• antibody
• epitope-focused
• immunization
• peptide
• vaccine

Since the time of Jenner, vaccines have prevented more morbidity and mortality than any other single medical intervention known to mankind.¹ Yet despite this success, a large burden of the disease-related morbidity and mortality today still occurs as a consequence of infections with malaria, tuberculosis, HIV, pandemic influenza, respiratory syncytial virus (RSV), dengue, S. aureus and other pathogens, all of which may be preventable through vaccination.²

Historically, vaccines have most commonly been live or attenuated viruses, heat-treated viruses, bacteria or bacterial supernatants, or sub-unit vaccines comprised primarily of pathogen-specific macromolecules. For many viral diseases including smallpox, polio and rabies, and bacterial pathogens like Bacillus anthracis, pneumococcus, and streptococcus, this approach has met with success. While these approaches may appear to lack some of the technological sophistication of some newer more molecular-based vaccine approaches, they have some distinct and long-standing advantages, including relative simplicity, high immunogenicity, and the elicitation of a typically redundant and broad antibody repertoire. Theoretically, the breadth of such a repertoire increases the likelihood that functional antibody will be elicited toward critical neutralizing epitopes, and compensates for individual and population variability in humoral responses. The approach to vaccination employing macromolecules or macromolecular complexes also has a high likelihood of effectively stimulating specific T cell immunity, that with the exception of T cell-independent antigens, is required for efficient priming of B cell responses, for the establishment of immunological memory and anamnestic immunity, and in the case of some pathogens, for the stimulation of protective cellular effectors.³

For some infectious agents, however, traditional approaches to vaccination may fail to protect, or even cause harm. The causes for such ineffectiveness are multifactorial and vary among the different pathogens, but commonly include the high mutability of protein targets in a pathogen, as is seen with HIV, and the lack of clearly-defined targets of protective humoral immunity, as is the case with S. aureus.⁴ Clearly, pathogens and pathogen-specific macromolecules are under selective pressure to evolve toward evasion rather than enhancement of immunogenicity, especially that directed toward critical neutralizing epitopes. For example, the preponderance of Ab elicited toward the hemagluttinin(HA)-containing seasonal influenza vaccine is directed toward the strain-specific hypervariable sequences. This in turn, necessitates the yearly development of new influenza vaccines which incorporate the HA from the most prevalent strains. Though conserved neutralizing sequences do reside in the stem region of HA, these sequences are immunologically quiescent in HA-containing vaccines.⁵ In HIV, neutralizing epitopes in envelope protein may be shielded or only uncloaked briefly during the infectious process such that antibiodies specific for these epitopes are ineffectively elicited by vaccines containing native HIV envelope protein.^6,7 For some viruses, such as RSV and the dengue virus, the elicitation of a broad repertoire of Ab against the virus may even be deleterious.^8,9

With the rise of reverse vaccinology,¹⁰ whereby genomic sequences are translated to define the complete antigenic repertoire of pathogens, and structural vaccinology,¹¹ which leverages the development of technologies capable of better defining the 3-dimensional structure of pathogen-specific macromolecules, we have dramatically increased our capability for specifically defining–and potentially targeting–the vast spectrum of pathogen-specific antigens. These approaches, combined with some of the more traditional techniques for identifying pathogen-specific neutralizing epitopes, including monoclonal Ab screening and mutational analysis, have led to what is often referred to as the “rational design” of vaccines. The ability to define, and then target individual and potentially critical pathogen-specific epitopes through immunization with epitope-focused immunogens is a potentially revolutionary approach–and indeed opportunity–in vaccinology. Some of the potential advantages of epitope-focused vaccination would include the ability to narrowly focus an immune response toward critical neutralizing epitopes while avoiding the elicitation of antibody specificities that play no role in protection, and may even be deleterious.¹² For example, the humoral response to the current protective antigen (PA)-containing anthrax vaccine induces a broad spectrum of antibody against PA, which is the principle antigen comprising Biothrax®, the approved anthrax vaccine in the US. Research has shown, however, that the vast majority of this antibody elicited to PA is likely non-neutralizing and is unlikely to play an important protective role in vaccinees.^13,14 Further, work in animal models has shown that some antibody specificities elicited following immunization with PA, or with lethal factor (LF), the second component of the A-B toxin which forms anthrax lethal toxin, may lead to antibody dependent enhancement (ADE) of disease in vitro and in vivo.^15-17 ADE has also been shown to occur in the context of traditional vaccination for a number of viral diseases including RSV and dengue.^8,18 Collectively, these data highlight one tradeoff associated with traditional immunization using modified pathogens or pathogen-specific macromolecules: they elicit very broad though often poorly characterized immunity. In many instances, including some of the great successes in modern vaccinology like smallpox, poliomyelitis and tetanus, such immunity elicits functional and protective immunity. For other pathogens, however, more focused and innovative approaches to vaccination may be required for the development of effective vaccines.

One approach to eliciting a more focused Ab response in vaccination is to subtract specificities from an otherwise broad repertoire through mutation or masking of undesirable epitopes.¹⁹ The alternative, and likely more applicable approach for widespread use, is to attempt to selectively elicit a highly-restricted Ab repertoire using an immunogen designed for this purpose: an epitope-focused vaccine. Conceptually, immunization with an epitope-focused vaccine may be thought of as the elicitation a mAb-like response against putative, or previously-validated neutralizing epitopes. The elicited response, of course, is not a monoclonal one but is indeed polyclonal. This itself is an advantage, enabling a more robust immune response capable of potentially accommodating mutations in the sequences comprising the neutralizing epitopes. Since the response is directed at only a delimited amount of sequence, typically in the form of linear peptide sequences, these vaccines can be very highly characterized with a high specific activity, or functional activity per mass of elicited antibody, and a low probability of ADE.^12,20 Typically, the development of such vaccines is predicated on eliciting Ab against linear neutralizing determinants. Depending of the formulation employed, which could include carrier-conjugated peptides, multiple antigenic peptides, peptides expressed on virus like particles or as part of other recombinant proteins, the effectiveness of such vaccines would typically be far less dependent on the elicitation of conformation-specific antibodies than immunization with intact macromolecules, and thus, would likely be less susceptible to denaturation and loss of activity compared to intact proteins, and attendant cold-chain requirements.

One especially unique attribute of epitope-focused vaccines is the ability to elicit antibody toward sequences that otherwise might not be immunogenic when vaccinating with attenuated, inactivated or killed pathogens, or pathogen-specific macromolecules. Indeed, we identified one such cryptic epitope in PA in the anthrax model, and a second cryptic epitope in α hemolysin of S. aureus.^21,22 In both cases, immunization with the full-length protein from which the epitopes were derived fails to elicit appreciable antibody to the neutralizing epitope in animal models, and for the PA epitope appears almost undetectable in human anthrax vaccinees as well. Yet immunization with epitope-focused immunogens was capable of eliciting protective levels of antibody against these cryptic epitopes in animal models of anthrax and S. aureus infections.^21,23,24 The identification of such immunorecessive epitopes has a parallel in the findings of the conserved, yet immunologically silent neutralizing epitopes in the stem region of HA, which one day may represent the focus of a new generation of influenza vaccines with the potential to obviate the requirement for annual influenza immunizations.^5,19

Like any new approach, however, many challenges remain in the development and ultimately, the application of epitope-focused vaccines. The loss of context of the target B cell from the native protein and from putative T cell epitopes, necessitates the provision of such T cell stimulation in the epitope-focused immunogen. Considerable research has been performed toward the identification of promiscuous helper T cell epitopes which can be effectively linked to B cell targets in epitope-focused vaccines, and while studies have demonstrated efficacy with these vaccines in inbred mice and rabbits, the application of this approach for human vaccination will require overcoming potential limitations imposed by MHC restriction.^25-28 Another long-standing approach for provision of such heterologous T cell stimulation is to covalently link the B cell target to a carrier protein like keyhole limpet hemocyanin, or as is the case with polysaccharide vaccines, to tetanus toxoid, detoxified diphtheria toxin (CRM197), or a meningococcal group B outer membrane protein.²⁹ An alternative approach with peptide epitopes is to express the target sequence as part of a larger protein like a virus like particle or a recombinant protein, which then provide the requisite T cell stimulation for priming and boosting the immune response.^12,20,30 Critical future research in this area is needed to better understand the potential and requirements for such epitope-focused vaccines to stimulate anamnestic immunity, especially in models like anthrax where this may be critical for protective efficacy.³¹ Though the B cell targets themselves may contain helper T cell epitopes capable of facilitating anamnestic boosting upon challenge with the pathogen, as has been shown in the rabbit model of anthrax, this is more likely the exception than the rule, and would provide inconsistent T cell help in a diverse population due to the requirements for MHC-restricted presentation.^23,32

Since the target B cell epitopes incorporated into epitope-focused vaccines are typically linear sequences of amino acids, one critique of this approach is that epitope-focused vaccines can only target simple, linear and contiguous neutralizing determinants.³³ While linear neutralizing determinants would appear to be the most logical target for this approach, recent work has shown that neutralizing Ab can be elicited to a conformational epitope in the F-protein of RSV through display of linear peptide sequences expressed on a virus like particle.³⁴ Other novel approaches for designing epitope-focused vaccines capable of targeting conformational or discontinuous neutralizing epitopes have also met with success.³⁵

Finally, the development, application and study of epitope-focused immunogens is not only conceptually attractive for diseases where conventional approaches to vaccination have met with obstacles, but this approach may also be uniquely suited for 2 other applications: the development of therapeutic post-exposure vaccines intended for pathogens where the rapid elicitation of antibody responses with mAb-like neutralizing specificities could mitigate disease severity, and importantly, for cancer therapy and prevention, where the elicitation of B and T cell responses toward tumor-specific antigens or antigens overexpressed in malignancy might prove efficacious.³⁶

Abbreviations

Ab	=	antibody
ADE	=	antibody-dependent enhancement
LF	=	lethal factor
PA	=	protective antigen
RSV	=	respiratory syncytial virus

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.