Combination vaccines: design strategies and future trends

Historically, the co-administration of vaccines to simultaneously immunize against multiple pathogens was motivated by a number of factors in clinical practice and advances in conjugate vaccine design. These factors include the requirement of certain immunizations in a given age bracket, epidemiologic overlap of certain infections, compatibility and stability of the vaccines, safety considerations, immunologic benefits and reduction of number of visits and injections. An additional stimulus for developing combination vaccines (CVs) is the availability of delivery vehicles that facilitates the simultaneous immunization against multiple diseases. In this respect, the technological advantage hinges on the capacity of a delivery system to accommodate antigens from different pathogens and target them effectively and safely to the immune system. In this editorial, we examine the major factors driving the development and expansion of CVs, with special emphasis on contemporary design strategies that include the use of novel delivery vehicles, such as virus-like particles, polycistronic DNA-mediated delivery, recombinant viral and bacterial vectors, and fusion proteins targeted to primary antigen presenting cells. Finally, other aspects of CVs are discussed in the context of viable areas for improvements and greater utility from this burgeoning vaccine strategy.

Vaccines offer a highly dependable strategy for preventing the mortality and morbidity of infectious diseases in a reliable and cost effective manner and, thus, constitute a vital tool in national and global public healthcare programs. Among the mounting vaccine success stories, the global eradication of smallpox, the significant worldwide suppression of paralytic poliomyelitis, tetanus, diphtheria and whooping cough, and the dramatic reduction in the incidence of Hemophilus influenzae type b (Hib) and Neisseria meningitidis are noteworthy [1]. It has been argued that the need for certain vaccine presentations will increasingly drive aspects of future vaccine development and manufacture. In this respect, vaccine presentations involving emphasis on single-dose preparations for combination vaccines (CVs) against multiple pathogens, and advances in vaccine delivery technologies to facilitate the design of such vaccines, will take center stage in vaccinology [2]. This editorial examines the evolving methodological and technological approaches to designing CVs and the potential impact of recent advances in novel vaccine delivery systems and carriers.

Definitions, requirements & benefits of CVs

Combination vaccines are either independent vaccine preparations that are combined during immunization or multiantigen constructs derived using a common carrier or delivery vehicle to design multiple subunit or conjugate vaccines against multiple pathogens [3,4]. There are several clinical, research and socioeconomic factors driving the steady expansion of strategies to design CVs. First, the increasing recommendations for national childhood immunization schedules to reduce infant and child mortality and morbidity owing to common infections has been a major factor. Thus, from just a few immunizations for diphtheria–tetanus–pertussis and measles–mumps–rubella during the first 2 years of life in the early 1980s, children under 5 years may presently receive up to 20 injections to complete their immunization series. These immunizations include doses of hepatitis B virus vaccine (HepB), a pneumococcal conjugate vaccine, inactivated poliovirus vaccine (IPV), influenza and HepA vaccines, in compliance with the recommendations of the American Academy of Pediatrics and the American Academy of Family Physicians [5,6]. The reality is that meeting these immunization schedules requires the use of CVs. For instance, the DTaP–HepB–IPV CV by GlaxoSmithKline Biologicals (Rixensart, Belgium) offers protection against five serious childhood diseases [5]. Currently available CVs and those in various stages of evaluation were recently reviewed [5,7–9]. Besides the traditional safety issues (e.g., microbial or chemical contamination and injection concerns) [2,10,11], the immunologic and biochemical effects of co-administering different vaccines (i.e., elicitation of adverse, suppressive, short- or long-term pathologic consequences) are other major considerations in developing CV. However, numerous preparations of CV have been medically certified and officially recommended as being sufficiently safe and effective for enhancing vaccination rates in children [5,8,12]. A primary requirement for endorsement of the use of CV is that any component of the combination is indicated and its other components are not contraindicated, and approval by the US FDA [7,8]. As an FDA requirement, a CV should exhibit immunogenicity comparable with the component vaccines and its safety profile should equal the most reactogenic component.

Secondly, the need to simultaneously target infections with epidemiological overlap in the population with a single-dose vaccine provides a basis for developing CVs. Furthermore, CV development takes advantage of the improvements in vaccine delivery systems, including the availability of delivery platforms with the capacity and stability to target multiple antigens to the immune system. Regardless of the presentation of CVs, those likely to rapidly earn widespread public healthcare acceptance should be heat stable, requiring no cold chain and freeze stable. Finally, future CVs could use a common route of administration to simultaneously target multiple pathogens with common sites of infection (e.g., agents of sexually-, oro-, gastric- or respiratory-transmitted diseases) or the simultaneous targeting of multiple diseases with one-dose vaccines for both therapeutic and prophylactic objectives.

Strategies for producing CVs

Traditional approaches for producing vaccines include the empirical attenuation of infectious agents (e.g., measles), chemical or physical inactivation of microbes (cellular pertussis or influenza virus) or bacterial toxins (tetanus and diphtheria) and recently biotechnological methods of genetic engineering for synthesizing highly purified antigens (e.g., hepatitis B virus) [1]. Regardless of the production method, it is crucial to preserve the relevant antigenic determinants that elicit immune responses recognizing the intact pathogen and absence of overt reactogenicity [13]. The correlates of protective immunity induced by a vaccine include the antimicrobial and neutralizing actions of specific antibodies, T cells and cytokines, although the extent of involvement of each effector may be different for different vaccines. Improved vaccine design will be enhanced by greater utilization of the accumulating knowledge in immunology, immunobiology and vaccinology. For instance, the immunologic concept of the carrier effect of proteins, discovered by Avery and Goebel, is utilized in developing conjugate vaccines, which involves linking an appropriate protein carrier to the carbohydrates and sugars that define the immunological specificities of these microbes and their serotypes (e.g., Hib, Meningococci and Pneumococci vaccines) [14]. In fact, improved knowledge of infectious agents’ immunobiology and promising findings in immunoregulation and immunomodulation have led to the discovery of more biologically relevant adjuvants and immunomodulators. These include the stimulators of innate immune response associated with Toll-like receptors and other cell surface molecules (CD40, Fc receptors, heat shock proteins, DC205, β-2 integrins and cytokine receptors), which promote the adaptive immunity induced by vaccines [15–17]. These advances will likely benefit CV design and use in the near future. Furthermore, the growing effort to produce novel vaccine delivery systems that can accommodate a multiple and seemingly limitless array of antigens offers a new opportunity for increased use of CVs to control or prevent diseases. The time-tested and promising approaches for producing CVs are discussed below and summarized in Table 1.

Production of pediatric CV by mixing individual vaccines or by conjugate vaccine production approach

CVs can be produced as admixtures of separately manufactured vaccines either in a licensed formulation or outside licensure, which was the earliest strategy for their production. Among other considerations, potential interactions between different components (antigens, adjuvants, buffers and preservatives) in the CV are ascertained, as well as the stability of the product [7]. A major impetus for applying the admixture approach is when the vaccines are delivered via the same route. In addition, the advent of conjugate vaccine design strategy provides a unique opportunity to design pediatric CVs by using a common protein carrier (shared component) on which microbial carbohydrates and other components are incorporated. Conjugate vaccines combine protein- or peptide-based antigens (e.g., diphtheria toxoid, tetanus toxoid, acellular pertussis, aP antigens, inactivated poliovirus strains, types 1, 2 and 3, and hepatitis B surface antigen) with a carbohydrate antigen(s) for enhancing immune responses against the latter portion(s) (e.g., Hib vaccine). Since several conjugate vaccines can use a common protein carrier, combination conjugate vaccines use the shared component to which microbial carbohydrates and other components are incorporated. For instance, in a vast majority of the CVs designed for pediatric use, the proteins in the toxoids DPaP are the common carriers or foundation on which other vaccines (e.g., Hib, HepB and IPV) are incorporated (e.g., the hexavalent formulation DTaP–IPV–Hib–HepB) [3]. Other CV preparations (e.g., measles–mumps–rubella) are also available or being evaluated [5,7]. Carrier-induced epitope suppression whereby pre-exposure to a high dose of the protein carrier in a conjugate vaccine can reduce the immune response to the conjugated epitopes, and antigenic competition involving epitopes on the same carrier competing for limited T-helper (Th) cells, are major issues in CV design [18–21]. To date, several combination conjugate vaccine developed for the pediatric population have provided the required safety, immunogenicity and efficacy, and this method of production is likely to remain in use until superior methods become available.

Other strategies for producing CV

Advances in vaccinology offer new delivery options for CV design, including mixtures of powered or liquid vaccines delivered under pressure using, for instance, the jet gun [22–24], oral administration of a mixture of vaccines in a liquid (e.g., the poliomyelitis vaccine), inhalation via aerosolization, plant-made vaccines delivered orally as heat-stable freeze-dried powder capsules and transdermally delivered vaccine mixtures using skin patches [2].

Potential future approaches to producing CV

The prospect of increasing the use of CVs is high but greater advances are required in constructing more suitable delivery systems for the multiple vaccines to be combined. In addition to the current focus on CVs for pediatric health, more CVs will be targeted to other populations, such as older people, populations facing special conditions (e.g., HIV-infected with multiple diseases) and noninfectious diseases, such as cancer cells with physiochemical or microbial (e.g., viral) etiologic agents. In this respect, the application of novel delivery systems will enhance progress in CV design. The ideal delivery systems for efficacious CVs should have the capacity to harbor multiple vaccine subunits, be safe and be administered via appropriate routes that promote the activation of a high level of the relevant immune effectors that control the targeted infectious agents or other diseases. Promising delivery systems that can be applied to CV design are listed as follows.

Bacterial delivery systems

Live, attenuated bacteria, such as the Lactobacillus[25], Salmonella and Listeria[26] systems, are promising delivery vehicles for CVs. In fact, as part of a normal vaginal flora and integrity, the generation of recombinant Lactobacillus expressing genes encoding multiple vaccine proteins for several sexually transmitted disease (STD) agents under a conditional promoter, will be a reliable CV strategy against multiple STDs. Also, nonliving bacterial delivery systems, such as recombinant bacterial ghosts, have the potential to direct the immune response against multiple antigens [27]. Bacterial ghosts are devoid of cytoplasmic contents while maintaining their native surface antigenic structures and cellular morphology. In the novel recombinant bacterial ghost vaccine strategy, multiple genes can be expressed on ghosts in a deliberately controlled manner to achieve high levels of an antigen or different antigens from the same or different pathogens, which can be presented to the immune system simultaneously as an effective combination or multicomponent vaccines against multiple agents [27,28]. Recent efforts in designing experimental recombinant Vibrio cholerae ghost (VCG) vaccines, by expressing select chlamydial and HSV-2 proteins in vibrios, induced protective mucosal immunity in a mouse genital infection model [29]. Recombinant VCG are safe, with relatively easy and cheap production, which offers a technological and manufacturing advantage for a CV required on a global scale.

Viral & DNA vectors

Vector-mediated immunization with naked DNA has received much attention, with delivery of several genes showing significant promise in animal and preclinical efforts [30–32]. Using a DNA or vector delivery strategy, genes encoding candidate CVs can be delivered as a polycistronic construct or fused with specific antigen presenting cell (APC)-targeting domains, such as the ligands for the costimulatory B7 [33], CD40 [34] or genes expressing specific cytokines [35]. Presently, the DNA vaccine strategy constitutes a highly useful tool for rapid screening for potential CV candidates in experimental models [36] pending the alleviation of DNA integration and toxicity concerns. Also, the use of recombinant viral vectors as delivery systems for CV deserves serious consideration. Noninfectious adenovirus [37], canarypox virus [38], vaccinia virus [39], respiratory viruses [40–43] and alphavirus replicons [44] are some of the well-characterized viral delivery systems with potential in CV design. For instance, a cold-adapted influenza vaccine expressing heterologous genes that encodes protective antigens of common STD agents (e.g., Chlamydia trachomatis, Neisseria gonorrheae, genital herpes, human papilloma virus, human papillomavirus [HPV] and HIV) can be administered as a nasal spray to simultaneously vaccinate against flu and these STDs. This is due to the cooperative interaction between the immune inductive sites of nasal-associated lymphoid tissues and the immune effector sites of genital mucosa, which are the infection sites of these STDs. Intranasal delivery of CVs against respiratory infection can combine the intranasal influenza vaccine with parainfluenza and/or respiratory syncytial virus vaccine. [40,43]. In addition, virus-like particles (VLP) are suitable for CV design since this delivery technology is reliable [45–47] and currently used in the newly FDA-approved HPV vaccine [48,49].

Cellular vaccine delivery

Immunization regimens involving the use of ex vivo antigen-pulsed or genetically engineered dendritic cells (DCs) to deliver and present antigens in vivo have produced promising experimental protective immunity against infectious diseases and cancers [50–56]. Antigen-pulsed DCs appear to possess the necessary antigenic, costimulatory and immunomodulatory features for inducing high levels of T-cell and antibody responses for optimal protective immunity. The practical application of vaccine delivery using DCs in adoptive immunotherapy in the regular clinical setting has been debatable. However, the phenomenal efficacy of the DC-based cellular vaccine makes them ‘natural adjuvants or pre-eminent delivery vehicles’ and useful tools in the design of effective delivery systems for immunizing against multiple infectious and noninfectious diseases and to unravel the necessary vaccine machinery in terms of antigens, delivery, immunity and homing requirements [57]. Therefore, the challenge for vaccinology is to develop a delivery system that will mimic the superior immunostimulatory properties of DCs to achieve more effective CVs.

Fc receptor-mediated delivery of fusion antigens to primary antigen-presenting cells

Fc receptor-mediated delivery of antigens, which are linked to the Fc portion of immunoglobulin (Ig) G, is an efficient method for targeting antigens to primary antigen-presenting cells (APCs) for the induction of a robust immune response without the need for exogenous adjuvant [58–60]. This process would allow recombinant fusion proteins defined from the protective antigens of multiple infectious agents and/or tumors to be targeted to DCs by linkage with the Fc portion of IgG.

Ensuring the design of more efficacious CVs

An improved understanding of the factors that control the generation and maintenance of substantial immunologic memory against CVs is crucial for the future of these reagents to confer long-lasting immunity against the various pathogens delivered simultaneously to the immune system. Thus, more studies are required to advance our understanding of:

•	The cellular and molecular basis of immunologic memory induced by the components of a CV, especially the role and properties of specialized cells (i.e., DCs, T and B cells), cytokines, in addition to other cellular and molecular elements of the innate and adaptive immunity

The immunoregulation of the effector function, migratory and homing properties, as well as the short- or long-term survival of naive (CCR7⁺) and mature or memory T cells, including the polarized Th1 (chemokine CC motif receptor [CCR5]⁺ and C-X-C chemokine receptor [CXCR]3⁺), Th2 (CCR3⁺, CCR4⁺ and CCR8⁺) cells in the effector memory (tissues) and central memory (lymph nodes) compartments of the immune system [61,62]. For example, targeted intermittent or periodic blockade of immune dampening molecules, such as program death-1 ligand and CTLA-4, on T cells may improve long-term memory, as recently suggested from studies involving CTLA-4 [63,64].

•

Considering the importance of CVs in infancy, an improved knowledge of neonatal immunocompetence in the areas of APC function of DCs, as well as strategies to modify the proclivity to induce a Th2 response [1]. Finally, since an important objective of vaccine combinition is to vaccinate against multiple diseases, the effect of multiple infections or multidisease conditions on immune responses against the individual infections should be better clarified.

Expert commentary

The initial impetus for developing CVs is centered on increasing immunization rates in the pediatric population to lower the morbidity and mortality of infectious diseases. However, CVs are also applicable to noninfectious diseases, in addition to any multidisease state in which individual disease can be controlled with vaccines. The expansion of the use of CVs will benefit from new delivery systems that can harbor multiple antigens and deliver them efficiently to the immune system. Potentially effective delivery systems for CVs should have the capacity to harbor multiple vaccine subunits, be safe and be administered via appropriate routes that promote the activation of high levels of the relevant immune effectors that control the targeted infectious agents or other diseases. Such delivery platforms can be used to target antigens against infections occurring at the same sites, such as STDs, oro–gastric and respiratory infection agents. Among others, promising effective delivery systems that are likely be used for CV design within the next 5 years include VLPs, polycistronic DNA-mediated delivery, recombinant live, attenuated and nonliving viral and bacterial vectors, and fusion proteins targeted to primary antigen presenting cells. Programmatic requirements for certain vaccine presentations by vocal interest groups, national and global immunization programs will constitute the vital forces to stimulate new delivery technologies that will greatly impact vaccine development and public availability [2]. Thus, a globally coordinated approach to defining vaccine priorities and an emphasis of research funding on CV delivery will promote the design of delivery technologies to advance CV production and use.

Table 1. Contemporary and future strategies for designing combination vaccine.

Design approach	Basis	Targeted disease(s)	Ref.
Admixtures of independent vaccines	Mixing separately manufactured vaccines in a licensed formulation or outside licensure.	Several pediatric vaccines	[7]
Incorporation of multiple vaccine subunits into a common protein carrier	A carrier protein used in several conjugate vaccines acts as a common vehicle for several vaccine subunits, such as carbohydrates and sugars.	Several pediatric vaccines in current use	[7]
Polycistronic DNA-mediated delivery	Coding sequences for protective antigens from multiple infectious agents are delivered as DNA immunization	Discussed in this review
Recombinant live-attenuated and nonliving viral/bacterial vectors; Virus-like particles (VLPs)	Recombinant live-attenuated or nonliving viruses and bacteria expressing protective antigens from multiple infectious agents	Discussed in this review
Fusion proteins targeted to primary antigen presenting cells	Recombinantly produced fusion proteins containing protective antigens from multiple infectious agents are targeted to dendritic cells via linkage with Fc portion of IgG	Discussed in this review
Cellular delivery of CVs	Use of dendritic cells pulsed with or engineered to express multiple antigens in adoptive cellular immunization	Discussed in this review

CV: Combinition vaccine; Ig: Immunoglobulin; VLP: Virus-like particles.

Acknowledgements

This work was supported by PHS grants (AI41231, GM 08248 and RR03034) from the National Institutes of Health and the Centers for Disease Control and Prevention (CDC). We appreciate the technical and editorial assistance and intellectual input of Deborah Lyn, Kahalia Joseph, Laurie Howard and Ila Churn.