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  1. 1. Microreviewcmi_1609 934..942 Optimizing vaccine development Daniel F. Hoft,1 * Vladimir Brusic2 and Isaac G. Sakala1 1 Division of Infectious Diseases, Allergy & Immunology, Saint Louis University School of Medicine, St. Louis, MO, USA. 2 Cancer Vaccine Center, Dana-Farber Cancer Institute, Boston, MA, USA. Summary Optimizing the development of modern molecular vaccines requires a complex series of interdiscipli- nary efforts involving basic scientists, immunolo- gists, molecular biologists, clinical vaccinologists, bioinformaticians and epidemiologists. This re- view summarizes some of the major issues that must be carefully considered. The intent of the authors is to briefly describe key components of the development process to give the reader an overview of the challenges faced from vaccine concept to vaccine delivery. Every vaccine re- quires unique features based on the biology of the pathogen, the nature of the disease and the target population for vaccination. This review presents general concepts relevant for the design and development of ideal vaccines protective against diverse pathogens. Topic 1: Antigen discovery There are many important considerations in the develop- ment of an effective vaccine (see Table 1), but the first issue in vaccine development is antigen choice. A vaccine must induce memory immune responses capable of rec- ognizing the intended vaccine target. These immune responses should be directed against highly conserved structures expressed by the pathogen. Use of highly con- served antigens in vaccines can minimize the chances of the pathogen achieving the immunological escape that can occur when hypervariable regions are used as vaccine antigens. These hypervariable regions rapidly accumulate mutational changes and provide wider diver- sity of immunological epitopes at the pathogen population level. Targeting required virulence factors can further focus vaccine responses on functional protection. Immu- nological destruction or neutralization of a key virulence factor in theory could render a pathogen innocuous even without preventing pathogen infection/replication. In addition to choosing antigens that are highly con- served and crucial for pathogen virulence, antigen choice requires considerations of the biology of the specific patho- gen to better predict which antigens could be ideal targets. For example, extracellular and intracellular pathogens are best targeted by antibody responses and T-cell responses respectively. For immunological protection against an extracellular pathogen, linear as well as conformational peptide, polysaccharide, glycopeptide and glycolipid epitopes expressed on the surface of the pathogen can provide targets for neutralization by high-affinity antibody responses. These high-affinity antibody responses can prevent pathogen infection and/or opsonize the pathogen for uptake and killing by professional phagocytes. The induction of optimal antibody responses normally requires help from CD4+ helper T cells. Therefore, vaccines designed to induce antibody responses protective against an extracellular pathogen should include both antibody epitopes and helper T-cell epitopes. Conversely, for an intracellular pathogen, the conven- tional ab T cells reactive with short peptide epitopes pre- sented by major histocompatibility complex (MHC) class I and II proteins on the surface of an infected cell are critical for protective immunity. T cells unlike antibodies can rec- ognize infected cells expressing short pathogen-derived peptides presented by MHC surface molecules. The acti- vated T cells can inhibit intracellular pathogen growth by: (i) production of cytokines capable of activating intracellular microbicidal activities, (ii) direct induction of infected cell apoptosis, or (iii) the release of cytolytic granules contain- ing perforin, granzymes and other components that can lead to cytolysis of the infected cells. The diversity of allotypic MHC alleles expressed by human populations, and the consequent variations in the relevant epitope specificity of protective T cells in different individuals, result in additional complexities regarding vaccine antigen choice for the development of T-cell vaccines designed to protect against intracellular pathogens. Combinations of Received 24 February, 2011; revised 4 April, 2011; accepted 7 April, 2011. *For correspondence. E-mail:; Tel. (+1) 314 977 5500; Fax (+1) 314 771 3816. Cellular Microbiology (2011) 13(7), 934–942 doi:10.1111/j.1462-5822.2011.01609.x First published online 2 June 2011 © 2011 Blackwell Publishing Ltd cellular microbiology
  2. 2. epitope targets that at least the majority of vaccine recipi- ents would be able to mount effective responses against must be selected. Recent advances in bioinformatics have led to the ability to predict T-cell epitopes with the capacity for widely promiscuous binding to multiple different common HLA types (‘superepitopes’ or ‘epibars’), which theoretically should be immunogenic in more than 90% of diverse populations (Meister et al., 1995; Sette and Sidney, 1998; Southwood et al., 1998; Wang et al., 2008; Gregory et al., 2009; Moise et al., 2011). Finally, antigen choice should take into consideration the principle of immunodominance.Avaccine needs to be able to induce potent immune responses that can easily recog- nize and act early during pathogen invasion. Naturally immunodominant T- and B-cell epitopes can provide effec- tive targets for protective immunity. In contrast, strongly immunodominant epitopes can prevent the development of more broadly protective immune responses, particularly important for immunity against a highly mutable pathogen. Furthermore, recent reports have suggested that some pathogens may have evolved to utilize immunodominance for their own advantage, in some cases to enhance the long-term survival of certain chronic pathogens in their hosts (Martin et al., 2006; Tzelepis et al., 2008). Thus, depending on the specific pathogen and how the pathogen has evolved with the host’s immune system, the inclusion of immunodominant epitopes could be beneficial or detri- mental for optimal vaccine efficacy. All of these critical issues for vaccine antigen selection discussed above must be carefully considered in the context of the specific pathogen being targeted. Topic 2: T helper subset differentiation and protective immunity The next issue for vaccine development is the need to determine the type of T helper (Th) subset needed to induce the relevant protective immune responses. CD4+ Th cells can differentiate into different effector subsets that produce distinct cytokine profiles characterized as Th1, Th2, Th3/regulatory T cells (Treg), Th17 and T folli- cular helper (Tfh) cells (Zhou et al., 2009; O’Shea and Paul, 2010). Th1 cells produce IFN-g, TNF-a and IL-2 important for activating intracellular microbicidal activities and for the generation of CD8+ cytolytic T lymphocytes (CTL), all of which are required for control of intracellular pathogens. Th2 cells produce IL-4, IL-5 and IL-13 which enhance antibody responses and antibody-dependent cellular responses required for control of extracellular pathogens. Th3/Treg cells produce TGF-b, IL-10 and/or IL-35, and are associated with potent secretory IgA responses protective against mucosally invasive patho- gens and minimization of immunopathology during chronic infections. Th17 cells produce IL-17, IL-21 and other cytokines/chemokines that enhance inflammatory recruitment of neutrophils and additional T cells, macroph- ages and dendritic cells. Th17 cells may be important for both direct control of extracellular pathogens along epi- thelial surfaces and indirect enhancement of protection against intracellular pathogens by recruitment of Th1 cells (Khader et al., 2007). Tfh produce IL-21 in germinal centres important for B-cell activation, differentiation and affinity maturation. Depending on the nature of the target pathogen, a vaccine may need to induce Th1 responses to protect against an intracellular pathogen, Th2 responses to protect against an extracellular pathogen, or Th17 and Th3 responses for protection against epithelial infection/ invasion. Specific differentiation factors have been identi- fied as critical for the generation of each Th subset. IL-12, IL-4, TGF-b, TGF-b plus IL-6, and IL-21 induce Th1, Th2, Th3, Th17 and Tfh respectively (Spolski and Leonard, 2010; Zhu et al., 2010). Each of these driving cytokines trigger distinct transcriptional activation programmes Table 1. Key considerations for development of a vaccine. Consideration Comments 1. Antigen discovery Conserved virulence factors, B/T epitopes depending on pathogen biology, immunodominance 2. Relevant CD4+ Th subset target Th1 versus Th2 versus Th17 depending on pathogen biology 3. Need for CD8+ T cells For intracellular pathogens, especially if replicates in cytoplasm and/or infects non-haematopoietic cells 4. Specific memory subset needed Central memory (Tcm) versus peripheral effector memory (Tpem) 5. Avoidance of excessive Treg Treg inhibit effector T-cell development but may be necessary for the generation of long-term memory 6. Adjuvant selection Alum salts enhance Ab responses; Toll-like receptor agonists, oil/water emulsions enhance T cells 7. Vectors/delivery format B- versus T-cell responses, ideally mimic natural pathogen invasion strategy 8. Schedules and routes of vaccination Mucosal versus systemic vaccinations depending on pathogen biology, heterologous boosters 9. Immunological networks/biomarkers Identify molecular signatures associated with optimal immune response, mucosal versus cutaneous trafficking 10. Phase I through III clinical trials Optimization of dosing, safety first, then immunogenicity and then protective efficacy Optimizing vaccine development 935 © 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 934–942
  3. 3. associated with differential master switches (e.g. Tbet, Gata3 and RORgt for Th1, Th2 and Th17 respectively). The Th1 and Th2 immune profiles may represent the most stable end-point phenotypes among these subsets, and have been shown to be programmable in long-term memory cells (Swain, 1994). In contrast, Th17, Th3/Treg and Tfh subsets may represent more transient, less dif- ferentiated states with more plasticity/reversion capacity (Zhou et al., 2009; O’Shea and Paul, 2010). The above mentioned differential driving cytokines, and other factors affecting the distinct transcriptional activation associated with these subsets, can be used to selectively induce the most relevant immune responses for the target pathogen (Hoft and Eickhoff, 2002). Additional subsets of T cells may be important targets for new vaccines, broadening the recognition potential of the immune response and/or facilitating the rapid recall of immunity. Certain T cells can recognize ceramides and other lipids presented by non-polymorphic CD1 mol- ecules, and can rapidly produce cytokine responses upon restimulation (Hiromatsu et al., 2002; Vincent et al., 2003). Similarly, the g9d2 TCR+ human T-cell subset can recognize phospholipids in an MHC-unrestricted fashion, can rapidly produce cytokines capable of enhancing Th1 immunity and can directly inhibit intracellular pathogen replication (Hoft et al., 1998; Shen et al., 2002; Morita et al., 2007; Spencer et al., 2008). Inclusion of key cera- mide and/or lipid antigens for specific activation of NKT and/or gd T cells may be important for the future develop- ment of some types of optimal vaccines. Topic 3: CD8+ T cells Conventional CD4+ T cells are stimulated by peptide epitopes presented by MHC class II molecules which generally sample extracellular antigens taken up by pinocytosis/phagocytosis, or antigens synthesized by pathogens that persist and/or replicate within the endoso- mal compartments of infected cells. CD8+ T cells are stimulated by foreign peptides presented by MHC class I molecules which sample cytoplasmic contents of infected cells. These CD8+ T cells contain cytolytic granules with perforin and granzymes responsible for membrane damage and apoptosis induction in infected target cells. Another molecular component of human cytolytic granules is granulysin which can mediate direct microbicidal activity against bacterial pathogens (Stenger et al., 1998). In addi- tion to this classical effector degranulation response with subsequent lysis and death of the recognized target cell, CD8+ T cells can trigger the inflammasome and/or lead to non-lytic effects that enhance intracellular suppression of certain pathogens like HSV (Knickelbein et al., 2008; Metkar et al., 2008). Because CD8+ T cells are generally stimulated by cytoplasmic peptides, vaccines that result in synthesis of antigens within the antigen presenting cell (e.g. live attenuated vaccine vectors and DNA expression vectors) are needed to induce optimal vaccine-specific responses. However, cross-presentation of soluble extra- cellular antigens by dendritic cells and B cells to CD8+ T cells can occur with the right adjuvant conditions (e.g. TLR signals that induce a ‘danger’ response including produc- tion of IL-12) (Bevan, 2006; Hoft et al., 2007). Topic 4: Memory T-cell subsets Different subsets of memory immune cells have been identified with complementary roles (Sallusto et al., 1999; 2004; Willinger et al., 2005). CCR7-expressing central memory T cells (Tcm) with high proliferative potential re-express lymph node homing receptors which allow for accumulation in lymph nodes. CCR7 negative, peripheral effector memory T cells (Tpem) are distributed throughout peripheral tissues, have little proliferative potential, but are able to rapidly provide protective effector functions at the initial peripheral sites of pathogen rechallenge. Tcm serve as efficient immune memory storage facilities waiting for dendritic cells from the periphery to bring early antigens upon remote pathogen rechallenge to the drain- ing lymph nodes where Tcm undergo restimulation, pro- liferative expansion and effector differentiation to dampen later waves of pathogen replication and spread. Tpem provide a first line of adaptive immune defence and gen- erally require persistence of antigen for their prolonged presence. Although still controversial, in general for long- term immune protection, the Tcm memory subset is prob- ably the best response to focus on inducing to provide optimal vaccine efficacy. However, experimental vaccines designed to maintain long-term induction of Tpem with chronically persisting vaccine vectors, with the goal of inducing optimal protection against initial epithelial inva- sion immediately upon pathogen exposure are being investigated (Hansen et al., 2009). In addition to proliferative expansion and effector T-cell function, the polyfunctionality of vaccine-induced memory immune responses is another important feature for con- sideration in vaccine development. For example, several studies have found that antigen-specific IFN-g production is not the only effector function important for optimal pro- tective immunity. Seder et al. demonstrated that the capacity of CD4+ T cells to produce all three Th1 cytok- ines, IFN-g, TNF-a and IL-2, was a much better predictor of the relative protective capacity provided by various leishmania vaccines in murine models than IFN-g produc- tion alone (Darrah et al., 2007). Similarly, CD8+ T cells capable of further proliferative expansion and production of both IFN-g and perforin were associated with delayed HIV disease progression while CD8+ T cells capable of producing only IFN-g were not (Migueles et al., 2002). 936 D. F. Hoft, V. Brusic and I. G. Sakala © 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 934–942
  4. 4. Consistent with these other results, we have shown that IFN-g production alone does not correlate with the ability of human T cells to inhibit intracellular mycobacteria (Hoft et al., 2002). Therefore, polyfunctional assessments of the memory T cells induced by vaccination are important for clinical development of the most effective vaccines. Topic 5: Treg Regulatory T cells are important for negative regulation of immunity, and the prevention of autoimmune diseases (Bluestone and Abbas, 2003). Natural and induced Treg develop in the thymus versus periphery, respectively, and can limit over-exuberant immune-mediated inflammation. Natural Treg express Foxp3, a master transcription factor that induces a detailed genome-wide molecule pro- gramme that maintains the Treg phenotype, and suppress effector T-cell responses through contact-dependent and -independent mechanisms. Induced Treg may or not express Foxp3, but still can inhibit effector T-cell responses through the production of IL-10 and/or TGF-b. Treg in general are critical for maintaining homeostasis of the immune system and minimizing the pathological effects of immune activation. However, many infectious pathogens induce Treg which can interfere with the devel- opment of optimal protective immunity. In fact, Belkaid et al. demonstrated that Treg can be responsible for the persistence of a chronic infection (Belkaid et al., 2002), indicating that some pathogens have learned to deliber- ately utilize the Treg response for their own advantage. Therefore, the efficacies of some vaccines and/or immu- notherapies directed against certain pathogens might be enhanced by limiting or actively inhibiting Treg responses. Furthermore, there is some evidence that certain T-cell epitopes can at least preferentially induce Treg rather than effector T-cell responses (Massa et al., 2007). Avoid- ing Treg biasing epitopes obviously could be important for the development of some types of vaccines. In addition to the natural and induced Treg discussed above, natural ligands produced in vivo during comple- ment activation can trigger signalling via CD46 on human T cells leading to negative feedback induction of IL-10 production in Th1 cells after peak immune responses are finished and no longer needed (Kemper et al., 2003; Cardone et al., 2010). Several pathogens have evolved to utilize CD46 as a specific target receptor for infection of host cells, and engagement between these pathogens and CD46 can trigger negative feedback inhibition of cell- mediated immune responses (Kemper and Atkinson, 2007). These negative regulatory effects triggered during initial infection can prevent the induction of optimal vaccine-induced immunity. We have shown that BCG immunity in humans is reduced by the effects of CD46 cross-linking on T cells, and natural ligands produced during BCG infection/replication can specifically enhance CD46 signalling (Truscott et al., 2010). Learning how to prevent these downregulatory signal- ling events may be important for learning how to induce more protective vaccine-induced immune memory. On the other hand, IL-10 produced by Treg has been implicated as a requirement for the induction of long-term memory, perhaps by inhibiting apoptosis in early effector T cells (Belkaid et al., 2002; Foulds et al., 2006). Therefore, further research efforts exploring the effects of Treg/ CD46-mediated regulation are necessary to optimize future vaccine strategies. Topic 6: Adjuvants Specific adjuvants can enhance immune memory and shape the phenotype of the recall response. Until very recently, the only adjuvants approved for human use were aluminium salts, which can increase the half-life of an antigen, improve uptake by professional phagocytes and trigger the inflammasome via NALP3 sensing (Eisenbarth et al., 2008). Alum adjuvants clearly increase the titres of specific antibodies generated by vaccination, but are not optimal adjuvants for the induction of CD4+ Th1 cell and CTL responses important for the control of intracellular pathogens. Newer adjuvants including oil/water mixtures with toll-like receptor triggering ligands (e.g. lipid A for TLR4, unmethylated CpG dinucleotide motifs for TLR9, etc.) are becoming available which can greatly enhance Th1 and CTL responses (Coffman et al., 2010). Classic immunological work has confirmed that Th1 and Th2 priming can induce stable differentiated phenotypes that maintain their original bias after remote recall stimu- lations (Swain, 1994). Systemic IL-12 administration (or administration of a TLR ligand such as a CpG oligonucle- otide that stimulates IL-12 production by dendritic cells and macrophages) during vaccination in animals can clearly bias for Th1/CTL response generation. Systemic IL-4 given during vaccination can skew for long-term Th2 memory immune cells. It is not clear yet whether Th3, Treg, Th17, NKT and/or gd T cells can develop similar stable long-term differentiated effector memory cells. However, driving cytokines have been identified for at least short-term induction of these latter immune subsets (e.g. TGF-b/IL-10 for Th3/iTreg generation; TGF-b plus IL-6, IL-21 and IL-23 for Th17 generation) (Zhu et al., 2010) and future research will focus on the importance of these additional subsets and their potential for attaining long-term stable immune memory phenotypes. Additional research will need to explore the relative values of using different membrane and cytoplasmic foreign sensors (TLR, RLR and NLR) individually and in combinations to induce the best vaccine-specific responses appropriate for each unique pathogen. Optimizing vaccine development 937 © 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 934–942
  5. 5. Topic 7: Vectors/delivery format Next for consideration are a diverse group of delivery systems all designed to result in antigens being synthe- sized within or translocated into the cytoplasm of antigen presenting cells in order to enhance T-cell stimulation and facilitate class I presentation to CD8+ CTL. Many viral and bacterial live attenuated vectors have been developed and shown to induce potent cell mediated immunity in animal models. These live attenuated vectors are highly immunogenic usually producing multiple pathogen- associated molecular patterns that signal through TLR/ RLR/NLR, providing the inflammatory signals and co-stimulation required to optimally induce protective immune responses. In addition, these live attenuated vectors can be delivered through more natural routes of pathogen invasion (e.g. through mucosal surfaces) poten- tially inducing more relevant regional immune responses for protection against the initial infection. However, by inducing potent inflammation these highly immunogenic live vectors can be associated with undesired reactoge- nicity, or may induce vector-specific immunity that can reduce the efficacy of booster vaccinations with the same vaccine. DNA vaccines, consisting of recombinant plasmids encoding vaccine antigens under the control of a eukary- otic promoter and containing TLR9 stimulatory CpG motifs, also deliver genes for expression inside the cyto- plasm of antigen presenting cells. These plasmid vaccine vectors have induced very potent Th1 and CTL responses in animal models but have not been as successful in humans to date. However, methods involving electropo- ration of plasmid into the superficial epidermis with an applied electrical current recently have been developed which result in enhanced targeting and uptake by resident Langerhan and other dendritic cells, and are likely to improve human DNA vaccinations (Hirao et al., 2011; Lin et al., 2011). Liposomal and cationic structures have been devel- oped that can allow translocation of soluble protein mol- ecules into the cytoplasm of antigen presenting cells. These additional delivery ‘vectors’ are potentially advan- tageous for mucosal vaccinations as these materials tend to be taken up by mucosal surfaces and concentrate within highly phagocytic cells (Heurtault et al., 2010; Mishra et al., 2010). This is a very active area of research currently, although so far there are no licensed vaccines consisting of liposomal/cationic formulated antigens. Topic 8: Schedules and routes of vaccination In addition to determining the right B- and T-cell epitopes, optimizing the capacity for selective induction of the appropriate immune subsets required, as well as selec- tion of the ideal adjuvant and delivery system, the sched- ules and routes of administration of each new vaccine must be optimized. For pathogens that invade through or are shed from mucosal tissues, mucosal vaccinations might induce optimal protection against initial infection and/or secondary transmission. Activation of T and B cells within immune inductive sites lining mucosal tissues leads to the upregulation of surface molecules on these cells important for allowing access to peripheral mucosal sites after circulation in blood. In fact, a network of mucosal immunity known as the common mucosal immune system results from the expression of mucosal homing molecules on immune cells activated in one mucosal tissue that can lead to enhanced distribution of these immune cells along most if not all mucosal tissues. Specific integrin com- plexes, additional adhesion molecules and chemokine receptors are involved in the specific dissemination of memory immune T and B cells to mucosa (Kunkel and Butcher, 2002). For example, the a4b7 integrin complex is upregulated on the surface of lymphocytes activated in the Peyer’s patches. This integrin specifically binds to MadCAM1 on endothelial cells and triggers transendothe- lial migration from the vasculature into the peripheral mucosal tissues. In contrast, intradermal or subcutaneous vaccinations induce cutaneous lymphocyte antigen (CLA) expression on T and B cells activated in the skin or lymph nodes receiving lymphatic drainage from cutaneous sites. CLA is important for recognition of molecular structures lining endothelial cells in cutaneous microcapillaries required for transpedesis into the peripheral cutaneous tissues. Therefore, by changing the route of a vaccination, regional immune responses relevant for protection against specific pathogens can be selectively enhanced. The optimal number of booster vaccinations must be determined for each new vaccine. Generally, more than 1 dose of a vaccine is required to induce optimal immune responses. Booster vaccinations lead to higher titre anti- body responses with increased affinity resulting from somatic hypermutation induced by recurrent stimulation within the hypervariable regions critical for antigen recog- nition. Two- to four-week intervals between booster vac- cinations historically have worked well for vaccines designed to induce protective antibody responses. Much less is known regarding the optimal boosting intervals for induction of Th1, CTL and other specific T-cell subsets. Although somatic hypermutation is thought not to occur in antigen-specific T cells, the most relevant clones are selectively amplified in response to booster vaccinations based on affinity reactions between MHC/peptide and TCR. These higher avidity T cells can lead to enhanced numbers of antigen-specific T cells, potency of effector responses and longevity of antigen-specific memory. Whether boosting T-cell responses after completion of their differentiation into maximal numbers of resting Tcm 938 D. F. Hoft, V. Brusic and I. G. Sakala © 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 934–942
  6. 6. could improve long-term T-cell immune memory is another important hypothesis to test. As mentioned above, vectors can induce vector- specific immunity that can limit the effectiveness of booster vaccinations with the same vaccine vector. Another important concept to investigate during vaccine schedule optimization is whether distinct molecular vaccine formats presenting the same vaccine antigens in heterologous prime/boosting combinations can enhance vaccine-induced protective memory immunity (Schneider et al., 1998; Wei et al., 2010). Priming with a DNA vaccine followed by boosting with a viral vectored vaccine, when both vaccines encode the same pathogen-specific anti- gens, can greatly enhance at least the total numbers of antigen-specific T cells that develop and persist long term. This technique of using heterologous prime/boosting schedules can overcome the problems encountered due to vector-specific immunity that develops during homolo- gous prime/boosting vaccinations, and may maximize the breadth and phenotype of immune subsets induced by providing T-cell stimulations in the context of wider com- binations of TLR/RLR/NLR. Topic 9: Immunological memory networks The ability to study whole genome-wide expression pat- terns in cells after different states of activation/ differentiation promises to revolutionize the way we approach vaccinology (Pulendran et al., 2010). Genome- wide expression comparisons of effector T cells and resting Tcm have demonstrated that the most potent effector cells represent the most terminally differentiated cells with the least capacity for self-renewal (Willinger et al., 2005). In contrast, Tcm are less terminally differen- tiated, have the best self-renewal/expansion capacity and because of increased expression of anti-apoptotic genes provide more long-term populations of antigen-specific memory T cells. Genome-wide expression pattern analyses have impli- cated basic metabolic pathways in effector versus memory T-cell generation. The mTOR pathway, activated by the presence of numerous substrates and important for growth and activation of primarily activated T cells, can bias for development of mostly short-lived effector T cells (Araki et al., 2009). Specific inhibitors of the mTOR pathway (e.g. rapamycin and metformin) can lead to increases in long-term memory T cells present after vac- cination. Further network analyses of immune cells have identified vitamin D metabolism as important for maximiz- ing the cutaneous trafficking potential of memory T and B cells, while vitamin A metabolism is involved in the induc- tion of mucosal trafficking potential (Sigmundsdottir and Butcher, 2008). The near future should bring additional genome-wide expression studies that identify gene net- works induced early on after vaccination that can predict optimal long-term protective immunity and characterize the more detailed factors involved in differential develop- ment of Th1/Th2/Th3/Treg/Th17 immune phenotypes and distinct lymphocyte homing programmes. To demonstrate the power of molecular transcriptomal analyses, we include preliminary data that we have recently generated studying human volunteers with previ- ous exposure to mycobacterial antigens. We have con- ducted a series of tuberculosis (TB) vaccine trials with the overall goal of learning how to improve the protective capacity of new TB vaccines. To our knowledge, there are no published reports of human T-cell antigen-specific molecular transcriptomes. Transcriptomes expressed in subsets of unactivated, total polyclonal human memory T-cell subsets have been studied, but not antigen-specific populations or the differences between rested and acti- vated memory T-cell responses. In mice, TCR transgenic models have facilitated studies of antigen-specific tran- scriptional profiles by providing highly purified populations of T cells that can be obtained with relative ease in naïve, activated effector and memory states. These pure popu- lations ensure all gene expressions being measured are related to the antigen-specific populations of interest. In humans, the frequencies of T cells specific for a given vaccine or pathogen usually are < 1–10% of the total T-cell population. Thus, it has been assumed that antigen- specific T cells must be purified in order to study complex gene expression pathways in a minority subset of antigen- specific T cells relevant for a given vaccine or infection. Practical issues with this approach include the small number of T cells recovered, the consequent need for amplification techniques to study the mRNA expressed, and potential alteration in gene expression profiles due to labour intensive in vitro manipulations. To address these feasibility concerns, we completed a pilot experiment which clearly demonstrates that TB-specific transcrip- tional profiles can be studied with a relatively simple approach not requiring purification of antigen-specific T cells. We detected BCG-induced changes in gene expres- sion among total memory CD4+ T cells (CD4+CD45RO+) purified from three PPD+ persons (VTR #1–3 in Fig. 1A). As shown in Fig. 1A, we detected greater than 50-fold increases in IL-2 mRNA at 24 h in T cells stimulated with BCG-infected compared with uninfected DC. These results demonstrate that we can detect marked increases in expression of the IL-2 target gene despite the fact that BCG-specific T cells likely represent only a minor fraction of all polyclonal CD4+ memory T cells, and indicate that stimulation of T cells for 24 h with BCG-infected DC (moi = 20) represent reasonable conditions for genome expression studies of BCG-specific T-cell responses. We prepared cDNA and cRNA from RNA samples harvested from CD4+ memory T cells co-cultured with uninfected Optimizing vaccine development 939 © 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 934–942
  7. 7. and BCG-infected DC for 24 and 48 h from these three volunteers and performed Affymetrix hybridizations with HG-U133 Plus 2 Affymetrix chips. The data were normal- ized and ANOVA used to identify genes with significant changes in expression comparing BCG-infected and unin- fected stimulation conditions. We found 3098 genes significantly altered by stimulation with BCG-infected DC and 608 upregulated at both the 24 and 48 h time points. Gene set enrichment analysis (GSEA) looking for networks of genes altered by BCG antigen-specific stimu- lation indicates that many gene sets classically associ- ated with immune response pathways (T-cell receptor signalling, Jak-Stat signalling, apoptosis, cytotoxicity, cytokine–receptor interactions and integrin-medicated cell adhesion) are highly represented among the significantly altered gene expression patterns (http://cvc.dfci.harvard. edu/share_folder/IMN). Therefore, we can use a relatively simple strategy to purify total memory CD4+ T cells and study the expression of a highly diverse set of human genes induced by antigen-specific stimulation. We are currently using this strategy to identify specific alterations in the antigen-specific gene set associated with mucosal versus cutaneous BCG vaccination, and which genes predict the best functional long-term memory responses after BCG vaccination. The overall goal is to determine new biomarkers that can be used to more rapidly and accurately assess mucosal and systemic immunogenicity of iterative TB vaccination approaches. Topic 10: Clinical development/safety and surrogate markers Once a vaccine is ready for clinical testing, the focus first becomes safety. Phase I dose escalation trials are designed to identify the safest and most immunogenic vaccine doses. Phase II trials expand safety analyses into larger numbers of volunteers and begin to address the efficacy of vaccination. Phase III trials are designed to provide sufficient statistical power to definitively address vaccine efficacy and include detailed reactogenicity assessments. Throughout the clinical development pathway from phase I to phase III, it is important to have surrogate markers of protective immunity that can be assessed in vaccinated volunteers. Ideally, correlates of protection should be known to help direct vaccine optimi- zation. Unfortunately, correlates of protection are not Fig. 1. Human molecular transcriptomal analyses can help identify important biological responses required for successful vaccines. In (A), memory CD4+ T cells were purified from three PPD+ persons with Miltenyi negative selection kits resulting in > 97% pure memory CD4+ T cells. Memory CD4+ T cells were stimulated with autologous DC (20:1 T : DC ratio) that were uninfected or infected with a BCG moi of 4, 20 or 100. Total RNA was harvested at 24, 48 and 72 h. We completed qRT-PCR for IL-2 mRNA to determine what conditions gave us the best ability to see BCG-induced changes. RNA harvested from the optimal conditions was used to identify the Affymetrix molecular signatures associated with BCG-specific stimulation demonstrating that the expression of 3098 genes was significantly involved (data not shown). (B) depicts the major phases of T-cell activation and the serial points of transcriptomal analyses being used to identify: (i) gene expression patterns involved in programming long-term protective immune memory, and (ii) the differential gene expression patterns that predict T-cell responses capable of providing optimal mucosal versus systemic immunity. 940 D. F. Hoft, V. Brusic and I. G. Sakala © 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 934–942
  8. 8. known for many of the human pathogens that remain key challenges for vaccine development. For HIV, TB and many other major world pathogens, only partial informa- tion regarding surrogates/correlates of protective immu- nity is available. This shortcoming makes iterative research critical involving the empirical development of vaccine candidates, clinical development of experimental vaccines and refinement of second-generation vaccines based on enhanced targeting of new surrogates/ correlates identified in vaccine trials with prototype vac- cines. Finally, once a vaccine is shown to be safe and efficacious in humans, additional research is necessary to assess ongoing effectiveness of the vaccine for preven- tion of infection and disease due to the target pathogen under real-world conditions, and to provide quality control for continual production of effective vaccines. Concluding remarks Vaccine development is a complex process involving mul- tiple different specialists, careful thought into the specific vaccine design, as well as laborious testing and evalua- tion. We have discussed some of the key issues important for the generation of a successful vaccine. Because of space limitations we have left out many additional steps that are necessary for this process including detailed animal testing of immunogenicity, protective capacity and toxicity. We hope that the reader now has a more com- plete appreciation of the detailed requirements for devel- opment of a successful vaccine. References Araki, K., Turner, A.P., Shaffer, V.O., Gangappa, S., Keller, S.A., Bachmann, M.F., et al. (2009) mTOR regulates memory CD8 T-cell differentiation. Nature 460: 108–112. Belkaid, Y., Piccirillo, C.A., Mendez, S., Shevach, E.M., and Sacks, D.L. (2002) CD4+ CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420: 502–507. Bevan, M.J. (2006) Cross-priming. Nat Immunol 7: 363–365. Bluestone, J.A., and Abbas, A.K. (2003) Natural versus adap- tive regulatory T cells. Nat Rev Immunol 3: 253–257. Cardone, J., Le, F.G., Vantourout, P., Roberts, A., Fuchs, A., Jackson, I., et al. (2010) Complement regulator CD46 tem- porally regulates cytokine production by conventional and unconventional T cells. Nat Immunol 11: 862–871. Coffman, R.L., Sher, A., and Seder, R.A. (2010) Vaccine adjuvants: putting innate immunity to work. Immunity 33: 492–503. Darrah, P.A., Patel, D.T., De Luca, P.M., Lindsay, R.W., Davey, D.F., Flynn, B.J., et al. (2007) Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major. Nat Med 13: 843–850. Eisenbarth, S.C., Colegio, O.R., O’Connor, W., Sutterwala, F.S., and Flavell, R.A. (2008) Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of alu- minium adjuvants. Nature 453: 1122–1126. Foulds, K.E., Rotte, M.J., and Seder, R.A. (2006) IL-10 is required for optimal CD8 T cell memory following Listeria monocytogenes infection. J Immunol 177: 2565–2574. Gregory, S.H., Mott, S., Phung, J., Lee, J., Moise, L., McMurry, J.A., et al. (2009) Epitope-based vaccination against pneumonic tularemia. Vaccine 27: 5299–5306. Hansen, S.G., Vieville, C., Whizin, N., Coyne-Johnson, L., Siess, D.C., Drummond, D.D., et al. (2009) Effector memory T cell responses are associated with protection of rhesus monkeys from mucosal simian immunodeficiency virus challenge. Nat Med 15: 293–299. Heurtault, B., Frisch, B., and Pons, F. (2010) Liposomes as delivery systems for nasal vaccination: strategies and out- comes. Expert Opin Drug Deliv 7: 829–844. Hirao, L.A., Draghia-Akli, R., Prigge, J.T., Yang, M., Satishchandran, A., Wu, L., et al. (2011) Multivalent smallpox DNA vaccine delivered by intradermal electropo- ration drives protective immunity in nonhuman primates against lethal monkeypox challenge. J Infect Dis 203: 95–102. Hiromatsu, K., Dascher, C.C., LeClair, K.P., Sugita, M., Furlong, S.T., Brenner, M.B., and Porcelli, S.A. (2002) Induction of CD1-restricted immune responses in guinea pigs by immunization with mycobacterial lipid antigens. J Immunol 169: 330–339. Hoft, D.F., and Eickhoff, C.S. (2002) Type 1 immunity pro- vides optimal protection against both mucosal and sys- temic Trypanosoma cruzi challenges. Infect Immun 70: 6715–6725. Hoft, D.F., Brown, R.M., and Roodman, S.T. (1998) Bacille Calmette-Guerin vaccination enhances human gd T cell responsiveness to mycobacteria suggestive of a memory- like phenotype. J Immunol 161: 1045–1054. Hoft, D.F., Worku, S., Kampmann, B., Whalen, C.C., Ellner, J.J., Hirsch, C.S., et al. (2002) Investigation of the relation- ships between immune-mediated inhibition of mycobacte- rial growth and other potential surrogate markers of protective Mycobacterium tuberculosis immunity. J Infect Dis 186: 1448–1457. Hoft, D.F., Eickhoff, C.S., Giddings, O.K., Vasconcelos, J.R., and Rodrigues, M.M. (2007) Trans-sialidase recombinant protein mixed with CpG motif-containing oligodeoxynucle- otide induces protective mucosal and systemic Trypano- soma cruzi immunity involving CD8+ CTL and B cell- mediated cross-priming. J Immunol 179: 6889–6900. Kemper, C., and Atkinson, J.P. (2007) T-cell regulation: with complements from innate immunity. Nat Rev Immunol 7: 9–18. Kemper, C., Chan, A.C., Green, J.M., Brett, K.A., Murphy, K.M., and Atkinson, J.P. (2003) Activation of human CD4+ cells with CD3 and CD46 induces a T-regulatory cell 1 phenotype. Nature 421: 388–392. Khader, S.A., Bell, G.K., Pearl, J.E., Fountain, J.J., Rangel- Moreno, J., Cilley, G.E., et al. (2007) IL-23 and IL-17 in the establishment of protective pulmonary CD4+ T cell responses after vaccination and during Mycobacterium tuberculosis challenge. Nat Immunol 8: 369–377. Knickelbein, J.E., Khanna, K.M., Yee, M.B., Baty, C.J., Kinchington, P.R., and Hendricks, R.L. (2008) Noncytotoxic Optimizing vaccine development 941 © 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 934–942
  9. 9. lytic granule-mediated CD8+ T cell inhibition of HSV-1 reactivation from neuronal latency. Science 322: 268– 271. Kunkel, E.J., and Butcher, E.C. (2002) Chemokines and the tissue-specific migration of lymphocytes. Immunity 16: 1–4. Lin, F., Shen, X., McCoy, J.R., Mendoza, J.M., Yan, J., Kemmerrer, S.V., et al. (2011) A novel prototype device for electroporation-enhanced DNA vaccine delivery simulta- neously to both skin and muscle. Vaccine (in press). Martin, D.L., Weatherly, D.B., Laucella, S.A., Cabinian, M.A., Crim, M.T., Sullivan, S., et al. (2006) CD8+ T-cell responses to Trypanosoma cruzi are highly focused on strain-variant trans-sialidase epitopes. PLoS Path 2: e77. Massa, M., Passalia, M., Manzoni, S.M., Campanelli, R., Ciardelli, L., Yung, G.P., et al. (2007) Differential recogni- tion of heat-shock protein dnaJ-derived epitopes by effec- tor and Treg cells leads to modulation of inflammation in juvenile idiopathic arthritis. Arthritis Rheum 56: 1648– 1657. Meister, G.E., Roberts, C.G., Berzofsky, J.A., and De Groot, A.S. (1995) Two novel T cell epitope prediction algorithms based on MHC-binding motifs; comparison of predicted and published epitopes from Mycobacterium tuberculosis and HIV protein sequences. Vaccine 13: 581–591. Metkar, S.S., Menaa, C., Pardo, J., Wang, B., Wallich, R., Freudenberg, M., et al. (2008) Human and mouse granzyme A induce a proinflammatory cytokine response. Immunity 29: 720–733. Migueles, S.A., Laborico, A.C., Shupert, W.L., Sabbaghian, M.S., Rabin, R., Hallahan, C.W., et al. (2002) HIV-specific CD8+ T cell proliferation is coupled to perforin expression and is maintained in nonprogressors. Nat Immunol 3: 1061–1068. Mishra, N., Goyal, A.K., Tiwari, S., Paliwal, R., Paliwal, S.R., Vaidya, B., et al. (2010) Recent advances in mucosal deliv- ery of vaccines: role of mucoadhesive/biodegradable poly- meric carriers. Expert Opin Ther Pat 20: 661–679. Moise, L., Buller, R.M.S., Schriewer, J., Lee, J., Frey, S., Weiner, D.B., et al. (2011) VennVax, a DNA-prime, peptide- boost multi-T-cell epitope poxvirus vaccine, induces pro- tective immunity against vaccinia infection by T cell response alone. Vaccine 29: 501–511. Morita, C.T., Jin, C., Sarikonda, G., and Wang, H. (2007) Nonpeptide antigens, presentation mechanisms, and immunological memory of human Vg9Vd2 T cells: discrimi- nating friend from foe through the recognition of prenyl pyrophosphate antigens. Immunol Rev 215: 59–76. O’Shea, J.J., and Paul, W.E. (2010) Mechanisms underlying lineage commitment and plasticity of helper CD4+ T cells. Science 327: 1098–1102. Pulendran, B., Li, S., and Nakaya, H.I. (2010) Systems vac- cinology. Immunity 33: 516–529. Sallusto, F., Lenig, D., Forster, R., Lipp, M., and Lanzavec- chia, A. (1999) Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401: 708–712. Sallusto, F., Geginat, J., and Lanzavecchia, A. (2004) Central memory and effector memory T cell subsets: function, gen- eration, and maintenance. Annu Rev Immunol 22: 745– 763. Schneider, J., Gilbert, S.C., Blanchard, T.J., Hanke, T., Robson, K.J., Hannan, C.M., et al. (1998) Enhanced immu- nogenicity for CD8+ T cell induction and complete protec- tive efficacy of malaria DNA vaccination by boosting with modified vaccinia virus Ankara. Nat Med 4: 397–402. Sette, A., and Sidney, J. (1998) HLA supertypes and super- motifs: a functional perspective on HLA polymorphism. Curr Opin Immunol 10: 478–482. Shen, Y., Zhou, D., Qiu, L., Lai, X., Simon, M., Shen, L., et al. (2002) Adaptive immune response of Vg2Vd2+ T cells during mycobacterial infections. Science 295: 2255–2258. Sigmundsdottir, H., and Butcher, E.C. (2008) Environmental cues, dendritic cells and the programming of tissue- selective lymphocyte trafficking. Nat Immunol 9: 981– 987. Southwood, S., Sidney, J., Kondo, A., del Guercio, M.F., Appella, E., Hoffman, S., et al. (1998) Several common HLA-DR types share largely overlapping peptide binding repertoires. J Immunol 160: 3363–3373. Spencer, C.T., Abate, G., Blazevic, A., and Hoft, D.F. (2008) Only a subset of phosphoantigen-responsive g9d2 T cells mediate protective tuberculosis immunity. J Immunol 181: 4471–4484. Spolski, R., and Leonard, W.J. (2010) IL-21 and T follicular helper cells. Int Immunol 22: 7–12. Stenger, S., Hanson, D.A., Teitlbaum, R., Dewan, P., Niazi, K.R., Froelich, C.J., et al. (1998) An antimicrobial activity of cytolytic T cells mediated by granulysin. Science 282: 121– 125. Swain, S. (1994) Generation and in vivo persistence of polar- ized Th1 and Th2 memory cells. Immunity 1: 543–552. Truscott, S.M., Abate, G., Price, J.D., Kemper, C., Atkinson, J.P., and Hoft, D.F. (2010) CD46 engagement on human CD4+ T cells produces T regulatory type 1-like regulation of antimycobacterial T cell responses. Infect Immun 78: 5295–5306. Tzelepis, F., de Alencar, B.C., Penido, M.L., Claser, C., Machado, A.V., Bruna-Romero, O., et al. (2008) Infection with Trypanosoma cruzi restricts the repertoire of parasite- specific CD8+ T cells leading to immunodominance. J Immunol 180: 1737–1748. Vincent, M.S., Gumperz, J.E., and Brenner, M.B. (2003) Understanding the function of CD1-restricted T cells. Nat Immunol 4: 517–523. Wang, P., Sidney, J., Dow, C., Mothe, B., Sette, A., and Peters, B. (2008) A systematic assessment of MHC class II peptide binding predictions and evaluation of a consensus approach. PLoS Comput Biol 4: e1000048. Wei, C.J., Boyington, J.C., McTamney, P.M., Kong, W.P., Pearce, M.B., Xu, L., et al. (2010) Induction of broadly neutralizing H1N1 influenza antibodies by vaccination. Science 329: 1060–1064. Willinger, T., Freeman, T., Hasegawa, H., McMichael, A.J., and Callan, M.F. (2005) Molecular signatures distinguish human central memory from effector memory CD8 T cell subsets. J Immunol 175: 5895–5903. Zhou, L., Chong, M.M., and Littman, D.R. (2009) Plasticity of CD4+ T cell lineage differentiation. Immunity 30: 646–655. Zhu, J., Yamane, H., and Paul, W.E. (2010) Differentiation of effector CD4 T cell populations. Annu Rev Immunol 28: 445–489. 942 D. F. Hoft, V. Brusic and I. G. Sakala © 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 934–942