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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Vaccine. Author manuscript; available in PMC 2013 July 31.
Published in final edited form as:
PMCID: PMC3729025

Technical Transformation of Biodefense Vaccines


Biodefense vaccines are developed against a diverse group of pathogens. Vaccines were developed for some of these pathogens a long time ago but they are facing new challenges to move beyond the old manufacturing technologies. New vaccines to be developed against other pathogens have to determine whether to follow traditional vaccination strategies or to seek new approaches. Advances in basic immunology and recombinant DNA technology have fundamentally transformed the process of formulating a vaccine concept, optimizing protective antigens, and selecting the most effective vaccine delivery approach for candidate biodefense vaccines.

Biodefense vaccines have been developed against a diverse group of pathogens. In this Vaccine supplement issue of “Biodefense Vaccines”, reviews are included to describe seven bacterial vaccines (Bacillus anthracis, Yersinia pestis, Francisella tularensis, Clostridium botulinum, Clostridium perfringens, Brucella, and Rickettsia), five viral vaccines (smallpox, Venezuelan equine encephalitis, influenza, hantavirus, and Rift Valley fever) and two additional viral vaccines against pathogens that cause diseases in animals (foot and mouth disease and blue tongue). For several pathogens, effective vaccines were developed in the past, such as those for smallpox, B. anthracis, Y. pestis, F. tularensis, and influenza. The discovery of vaccination to prevent smallpox in 1796 by Edward Jenner actually marked the start of vaccinology [1]. The smallpox vaccine was so successful that it eventually eradicated this disease completely from the human population. However, when vaccines need to be produced for purposes of biodefense in the 21st century, the challenges appear greater than we would have expected. While governments of developed countries are willing to allocate many billions of dollars for the production and stockpiling of biodefense vaccines to protect the public from a potential bioterrorism attack, there appears to be a bottleneck for actual production of these vaccines, as evidenced by the vaccines on the government's procurement list that still have yet to be produced.

The reasons for drastic changes over the past 200 years to produce the same smallpox vaccines deserve more review; however, one of the key factors is the continuous and significant change on the social attitude and government regulatory controls regarding the safety standards for vaccines, as part of the broad medical products. Modern concepts of good manufacturing practice (GMP) basically disqualify many vaccines developed and produced during pre-GMP time as human vaccines. “Efficacy first” has become “safety first” during regulatory review of candidate vaccines. At the same time, the definition of safety and society's tolerance towards the risks associated with vaccines are also evolving. Mortality, which was rare even with the old vaccines, can no longer be viewed as the only valid safety standard. Rare vascular [2, 3] or autoimmune findings [4] can halt a vaccine clinical trial along its development pathway. Even diseases of low frequencies and unknown linkages to vaccines can lead to the complete withdrawal of a vaccine, even after its licensure [5]. While not all of these examples are directly related to the development of vaccines for purposes of biodefense, the development of biodefense vaccines also faces the same regulatory environment. It is important to stress that the intention and practice of more stringent regulatory review are a reflection of society's demands and, as a result, it is not fair to simply blame a regulatory agency or any administrations of a particular government for making the development of vaccines more difficult. Rather, it falls upon researchers in the field of vaccinology to advance vaccine technology to meet increasingly higher regulatory requirements in addition to the need to identify immunogens and exploit them for vaccine development.

Significant progress in immunology has provided vaccinologists with increased knowledge of human vaccines. At the same time, deeper knowledge in the theory and practices of modern-day immunology has created a discord between empirical and rational approaches for vaccine development. Frequently, the empirical approach will be labeled as “not hypothesis driven”. The scientific community is no longer satisfied with a candidate vaccine unless detailed and highly specific hypotheses can be provided on its possible mechanisms of protection and efficacy. As a result, the development of new generation biodefense vaccines requires both immunogenicity and mechanistic studies to provide more complete description and justification for the candidate vaccines, which is a significant change from past-generation vaccines.

No doubt, the development and production of biodefense vaccines in the 21st century is experiencing a fundamental technology transformation to meet new challenges. The following sections provide a brief overview of this movement (Table 1).

Table 1
Technological issues facing the development of biodefense vaccines

Changes in manufacturing processes

Anthrax bioterrorism attack that ensued in the U.S. shortly after September 11, 2001 was the key turning point in the history of biodefense vaccine development. Suddenly, the need for biodefense vaccines became eminent and moved from the traditional military market (i.e. protecting soldiers from biological weapons) to one incorporating both military and public use. However, many problems associated with the production of early generations of biodefense vaccines were also exposed. Vaccines for smallpox and anthrax provided two typical examples.

The traditional smallpox vaccines were produced by infecting the skin of calves or other large animals. The infected areas were then scraped to collect the lymph exudate, and live attenuated virus was purified and stabilized as human vaccines [1]. Such production continued until the 1970-1980s. In the early 2000's, when a new stock of smallpox vaccine was needed, it was clear that the antiquated and primitive manufacturing process for this vaccine was no longer acceptable for modern-day safety standards.

For the available anthrax vaccines, avirulent bacterial culture extracts are produced and the secreted protective antigen (PA) formed the basic component of Anthrax Vaccine Absorbed (AVA). AVA was licensed in 1970s but its release for routine use was only approved after the manufacturers provided supplemental information related to the manufacturing process in addition to a change in the label and package insert in 2002. As noted by the US General Accounting Office in 2002 during its review of existing anthrax vaccines, “general public health vaccines are produced according to cGMP and are in constant, routine use worldwide. This use permits real time monitoring of whether the vaccines are performing properly. In contrast, biodefense vaccines have no such ongoing reality check because of the absence of natural disease and relatively limited use” [6].

Since then, biodefense vaccines no longer receive special treatment. The same stringent FDA review, based on cGMP requirements, is now applied to vaccines developed for the purpose of biodefense as is required for other, more routine human vaccines. A new version of the smallpox vaccine was produced by using the cell culture method, a major technical advance from its original production in live animals. This new smallpox vaccine, ACAM2000, was licensed in 2007 and later selected for national stockpile in the U.S [7].

However, other biodefense vaccines have not been able to make similar rate of progress in the manufacturing process (Table 2). A recombinant PA protein-based anthrax vaccine has not advanced fast enough to replace AVA, although, the PA protein vaccine was initially considered a relatively easy vaccine candidate [8]. Many biodefense vaccines are currently under development at various smaller biotechnology companies that lack significant experience in vaccine development and manufacturing when compared to the larger pharmaceutical companies. Some of them rely on limited product pipeline and usually cannot invest significantly in more complicated manufacturing process. The big pharmaceutical companies did not appear to be interested in biodefense vaccines considering that the only potential customers for biodefense vaccines are government agencies who can revise procurement policies or require new paperwork at any time with limited interest in compensating the extra cost associated with early research and development effort of biodefense vaccines.

Table 2
Candidate vaccines against key bioterrorism agents

Additional features of biodefense vaccines have made the process of producing biodefense vaccines even more challenging. The requirement of an extended shelf-life, for stockpiling reasons, is one such complication. Ideally, a biodefense vaccine should be expected to preserve the high levels of efficacy after being stockpiled for at least 3-5 years and possibly longer. Due to the high cost associated with stockpiling of vaccines, benefits of producing biodefense vaccines that can withstand extended periods of shelving are evident. This is a major challenge to the current vaccine manufacturing process since most routine vaccines do not need to be stored for extended periods of time, and, as a result, are more often produced for use in the near future due to cost and quality control issues.

Another issue underlying the development of biodefense vaccines is that the main pathway of transmission for several lethal bioterrorism pathogens is considered to be aerosol-based. Although there are new aerosol technologies to deliver vaccines, they would require a redesigning of the manufacturing process to formulate vaccines to match the downstream aerosol delivery equipment as well as the viability of the product due to humidity and temperature [9].

Adjuvant is also receiving significant attention in biodefense vaccines because it has become clear that not only the subunit vaccines, but also the inactivated vaccines, will need adjuvants to be part of a highly immunogenic vaccine formulation [10]. The complex nature of adjuvant-vaccine formulation presents an additional challenge in biodefense vaccine development (see below).

Reverse genetics approach used in influenza vaccine production improved a key step in the manufacturing of influenza vaccines, i.e., the generation of seed strain viruses that are used to produce both inactivated and live attenuated influenza vaccines [11, 12]. Reverse genetics can not only speed up the generation of the typical reassortant seed strains to seasonal and low pathogenic influenza viruses but also produce a safe seed strain for the highly pathogenic H5 or H7 strains because the molecular features of HA antigen which confers the high virulence of these viruses can be removed during the process of reverse genetics, improving the safety of manufacturing vaccines for highly pathogenic influenza viruses.

Correlates of immune protection

Traditionally, it has been postulated that vaccines are effects through the induction of protective antibodies in the host. In recent years, with the availability of more sophisticated biomarker assays that were initially developed for routine human vaccines, the roles of T cell immune responses have been better recognized in biodefense vaccine studies.

One surprising finding came from plague vaccine studies. Smiley and colleagues showed that B cell-deficient mice vaccinated with live attenuated Y. Pestis were protected against plague while depleting T cells at the time of challenge abrogated protection and transferring vaccine-primed T cells to naïve mice provided protection [13]. These results established that cellular immunity mediated by vaccine-primed T cells can indeed protect against plague.

By using a mouse model, Berzofsky's group demonstrated that although antibody was essential to protect against disease by smallpox vaccines, T cells were necessary and sufficient for survival and recovery in the absence of protective antibodies [14]. Furthermore, biodefense vaccine-specific T cell immune responses can be present in immunized human populations for a long period. Demkowicz et al. showed that long-lived vaccinia virus-specific memory cytotoxic T cells were present in adults who had been immunized against smallpox as children. In people who had been immunized 35-50 years earlier, significant CD8+ and CD4+ T cell responses to vaccinia virus were detected after in vitro stimulation while no such responses were detected in young adults with no history of immunization against smallpox [15].

The above studies challenge us to re-evaluate the traditional way of thinking on how biodefense vaccines may work, which is important since some previous generation biodefense vaccines have been widely used for many years. Identification of the role of T cell immune responses with biodefense vaccines raised the question of what the correlates of protection for candidate biodefense vaccines may be. In many cases, there is no gold standard to make such judgment. It becomes even more complicated that for some biodefense vaccines, both antibody and T cell immune responses may be important for protection. At this point, it is unclear whether any successful biodefense vaccine will require strong responses for both arms of immune system. A key conceptual challenge is encountered when one takes into account that protective antibodies may be effective at the time of pathogen invasion, or an anamnestic response can be quickly induced to produce high level antibodies to eliminate or limit the infection, while it may take days to produce an antigen-specific T cell immune response, which may not be soon enough to prevent the pathogen from establishing an infection. In general, T cell responses are mainly effective against infected cells but not cell-free pathogens. The answer to these questions may depend upon the individual pathogen or biodefense vaccine in question, however, issues surrounding immune correlates of protection should be the first critical step to consider in the design of biodefense vaccines.

A related issue is the development of validated biomarker assays using good laboratory practice (GLP) and appropriate standards to measure the particular component of immune responses as the correlates of protection. In the case of cell mediated immunity, significant progress in recent years has been made in developing more quantitative and reproducible assays such as ELISPOT and intracellular cytokine staining with human peripheral PBMCs. Novel antibody assays are also under rapid development. New assays for detecting protective antibodies, such as the use pseudotyped viruses, will provide functional measurements in addition to traditional ELISA-based antibody assays. B cell ELISPOT may provide more information on the memory B cell status. Highly sensitive and quantitative solid phase-based assays can now detect multiple antigen specific antibodies at even very low levels. The combination of these biomarker assays will contribute to the identification of correlates of protection for future generations of biodefense vaccines.

Subunit protein or gene-based vaccination approaches

Traditional biodefense vaccines were designed by using either live attenuated or inactivated vaccine approaches. As discussed in the above sections, live attenuated vaccines have the benefit of normally inducing stronger protective immunity than inactivated vaccines but are not ideal candidates when facing the challenges related to manufacturing processes or more critically, to improve the overall safety profile of biodefense vaccines. Furthermore, regulatory authorities expect to see the mechanism of attenuation to be well-characterized. On the other hand, inactivated vaccines, although safe in general, are usually not very immunogenic, require multiple immunizations to reach the protective levels of immunity, and have not been shown to be good inducers of T cell immunity when used alone.

Given that it takes 18-20 years to develop a vaccine in many cases, there are major initiatives to develop new platform technologies where selected protective antigens, or genes for such antigens, but not the whole pathogen as in the cases of live attenuated and inactivated vaccines, can be incorporated to generate a vaccine against a biodefense agent at short notice. At the present time, there is still a long way to achieve this goal but there are a number of promising platform technologies.

In the near term, recombinant protein-based vaccines are attractive alternatives because they can be produced using a highly standardized manufacturing process, are safer than using the entire pathogen (either live or inactivated), and should be no less immunogenic than inactivated vaccines if a proper adjuvant is included in the final formulation. Examples of the successes of such platform technology in the public health arena are hepatitis B virus (HBV) and human papillomavirus (HPV) vaccines. Recombinant protein-based vaccines represent several forerunners of the newer generation of biodefense vaccines, especially for bacterial pathogens with well-characterized protective antigens. For plague vaccines, recombinant proteins F1 and V have been well-established as key protective antigens and vaccines based on these proteins have entered clinical studies [16]. Vaccines based on the recombinant protein PA antigen have been the leading candidate for a newer generation of anthrax vaccines [8]. For certain toxin-producing bacterial pathogens, recombinant protein-based subunit vaccines based on modified toxins are also replacing the traditional toxoid as the protective antigen, such as in the case of Botulinum vaccines [17]. Despite the advantages for recombinant protein vaccines, one key weakness for subunit-based vaccines is their poor immunogenicity for T cell responses.

Gene-based vaccines, on the other hand, have emerged in the last decade as a completely novel strategy for vaccination [18, 19]. At first, their ability to induce antigen-specific T cell responses was considered as the main strength. Over time, however, it became clear that gene-based vaccines are also effective in eliciting antibody responses. Gene-based vaccines include DNA vaccines and vector-based vaccines. Vectors can be either viral or bacterial, but more biodefense vaccines use viral vectors over bacterial vectors. Although both DNA vaccines and vector-based vaccines incorporate a natural or modified gene from a pathogen, which encodes the protective antigen, they differ in many ways. First, DNA vaccines can be delivered directly in the form of plasmids whereas the vector approach usually required the production of a large stock of highly concentrated, packaged vector vaccines. Second, most DNA vaccines generally do not contain unrelated proteins in the construct. The vaccinated hosts will only generate immune responses against the biodefense antigen expressed by the DNA vaccines. In contrast, vector-based vaccines express other antigens as part of the original vector virus or bacteria. Immune responses against vector components can generate several negative effects. For hosts who have been exposed to the same vector in the past, such as in the case serotype 5 of adenovirus (Ad5), pre-existing immune responses against the Ad5 can reduce the ability of a vaccine using Ad5 vector as the delivery system [20]. Anti-vector immunity may also interfere with protection of vector-based vaccines, as was observed during the STEP trial, a large international HIV vaccine clinical trial co-sponsored by the National Institute of Allergy and Infectious Diseases and the pharmaceutical company Merck & Co. Inc., in which people with high pre-existing anti-Ad5 antibody responses had a higher chance of being infected by HIV-1 when these people were immunized with an Ad5-based HIV-1 vaccine [21].

As shown in Table 2, the DNA vaccine approach has been tested for almost every biodefense pathogen due to the relatively simple nature of this approach. Vector-based vaccines have also been tested for many biodefense pathogens. We have included in Table 2 key references of published work on these gene-based vaccination approaches for each of the key pathogens included in the current Supplement. Most of these studies were conducted in small animal models and were successful in eliciting positive immunogenicity results against these biodefense pathogens, and some obtained data on protection in the cases where validated animal models exist.

The key information missing for many of these studies is the comparison between gene-based vaccines and other vaccines using traditional approaches. Such comparison would provide valuable information on the actual improvement of immunogenicity with these gene-based vaccination approaches over the existing and imperfect vaccines. However, the main challenge for gene-based vaccines is the overall low immunogenicity demonstrated in human studies for these vaccines [22]. But significant progress has been made in at least three key areas in recent years to address this issue. Molecular adjuvants, in the form of genes coding for immune-stimulating cytokines, have been shown to significantly improve the overall immunogenicity of DNA vaccines in non-human primate models [23, 24]. Physical delivery approaches, such as gene gun and electroporation, were shown more effective in eliciting higher levels of immune responses than the traditional needle injection method [25, 26]. Finally, the prime-boost strategies in which DNA or vector based vaccines were used in combination with another form of vaccines, such as in the case of DNA prime-protein boost [27], or DNA prime-Ad5 vector boost [28], have shown real promise in eliciting high and balanced antibody and T cell immune responses in both animal and human studies.

Future outlook

Since 2001, biodefense vaccine development has experienced significant progress in the areas of manufacturing, immunological mechanisms and novel vaccination approaches. There are other unique issues not covered in this technology review but they are equally important for biodefense vaccine development. These include 1) “animal rule” which applies to vaccines unable to conduct a late phase large scale efficacy trial in humans due to the rare natural occurrence of such infections; 2) bio-engineered threats which constitute a unique bioterrorism threat not belonging to the traditional pathogens; and 3) rules and process governing the use of IND (investigational new drug) status vaccines which can be developed and stockpiled for Emergency Use Authorization (EUA) purpose as established by the Project BioShield Act of 2004 (Public Law 108-276). Future successful biodefense vaccines need to be innovative to satisfy increasingly demanding requirements in safety and efficacy. It is less likely that one format will fit every biodefense vaccine, but the future successful biodefense vaccines no doubt will incorporate many technological innovations.


Authors are supported in part by NIH NIAID grant U01AI078073.


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