vacunasoptimas

Microreviewcmi_1609 934..942

Optimizing vaccine development

Daniel F. Hoft,1* Vladimir Brusic2 andIsaac G. Sakala1

1Division of Infectious Diseases, Allergy & Immunology,Saint Louis University School of Medicine, St. Louis,MO, USA.2Cancer Vaccine Center, Dana-Farber Cancer Institute,Boston, MA, USA.

Summary

Optimizing the development of modern molecularvaccines 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 thatmust be carefully considered. The intent of theauthors is to briefly describe key components ofthe development process to give the reader anoverview of the challenges faced from vaccineconcept to vaccine delivery. Every vaccine re-quires unique features based on the biology of thepathogen, the nature of the disease and the targetpopulation for vaccination. This review presentsgeneral concepts relevant for the design anddevelopment of ideal vaccines protective againstdiverse pathogens.

Topic 1: Antigen discovery

There are many important considerations in the develop-ment of an effective vaccine (see Table 1), but the firstissue in vaccine development is antigen choice. A vaccinemust induce memory immune responses capable of rec-ognizing the intended vaccine target. These immuneresponses should be directed against highly conservedstructures expressed by the pathogen. Use of highly con-served antigens in vaccines can minimize the chances ofthe pathogen achieving the immunological escape thatcan occur when hypervariable regions are used asvaccine antigens. These hypervariable regions rapidly

accumulate mutational changes and provide wider diver-sity of immunological epitopes at the pathogen populationlevel. Targeting required virulence factors can furtherfocus vaccine responses on functional protection. Immu-nological destruction or neutralization of a key virulencefactor in theory could render a pathogen innocuous evenwithout preventing pathogen infection/replication.

In addition to choosing antigens that are highly con-served and crucial for pathogen virulence, antigen choicerequires considerations of the biology of the specific patho-gen to better predict which antigens could be ideal targets.For example, extracellular and intracellular pathogens arebest targeted by antibody responses and T-cell responsesrespectively. For immunological protection against anextracellular pathogen, linear as well as conformationalpeptide, polysaccharide, glycopeptide and glycolipidepitopes expressed on the surface of the pathogen canprovide targets for neutralization by high-affinity antibodyresponses. These high-affinity antibody responses canprevent pathogen infection and/or opsonize the pathogenfor uptake and killing by professional phagocytes. Theinduction of optimal antibody responses normally requireshelp from CD4+ helper T cells. Therefore, vaccinesdesigned to induce antibody responses protective againstan extracellular pathogen should include both antibodyepitopes 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 Iand II proteins on the surface of an infected cell are criticalfor protective immunity. T cells unlike antibodies can rec-ognize infected cells expressing short pathogen-derivedpeptides presented by MHC surface molecules. The acti-vated T cells can inhibit intracellular pathogen growth by: (i)production of cytokines capable of activating intracellularmicrobicidal activities, (ii) direct induction of infected cellapoptosis, or (iii) the release of cytolytic granules contain-ing perforin, granzymes and other components that canlead to cytolysis of the infected cells. The diversity ofallotypic MHC alleles expressed by human populations,and the consequent variations in the relevant epitopespecificity of protective T cells in different individuals, resultin additional complexities regarding vaccine antigenchoice for the development of T-cell vaccines designed toprotect against intracellular pathogens. Combinations of

Received 24 February, 2011; revised 4 April, 2011; accepted 7 April,2011. *For correspondence. E-mail: [email protected]; 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.xFirst published online 2 June 2011

© 2011 Blackwell Publishing Ltd

cellular microbiology

epitope targets that at least the majority of vaccine recipi-ents would be able to mount effective responses againstmust be selected. Recent advances in bioinformatics haveled to the ability to predict T-cell epitopes with the capacityfor widely promiscuous binding to multiple differentcommon HLA types (‘superepitopes’ or ‘epibars’), whichtheoretically should be immunogenic in more than 90% ofdiverse populations (Meister et al., 1995; Sette and Sidney,1998; Southwood et al., 1998; Wang et al., 2008; Gregoryet al., 2009; Moise et al., 2011).

Finally, antigen choice should take into consideration theprinciple of immunodominance.Avaccine needs to be ableto induce potent immune responses that can easily recog-nize and act early during pathogen invasion. Naturallyimmunodominant T- and B-cell epitopes can provide effec-tive targets for protective immunity. In contrast, stronglyimmunodominant epitopes can prevent the developmentof more broadly protective immune responses, particularlyimportant for immunity against a highly mutable pathogen.Furthermore, recent reports have suggested that somepathogens may have evolved to utilize immunodominancefor their own advantage, in some cases to enhance thelong-term survival of certain chronic pathogens in theirhosts (Martin et al., 2006; Tzelepis et al., 2008). Thus,depending on the specific pathogen and how the pathogenhas evolved with the host’s immune system, the inclusionof immunodominant epitopes could be beneficial or detri-mental for optimal vaccine efficacy.

All of these critical issues for vaccine antigen selectiondiscussed above must be carefully considered in thecontext of the specific pathogen being targeted.

Topic 2: T helper subset differentiation andprotective immunity

The next issue for vaccine development is the need todetermine the type of T helper (Th) subset needed to

induce the relevant protective immune responses. CD4+Th cells can differentiate into different effector subsetsthat produce distinct cytokine profiles characterized asTh1, Th2, Th3/regulatory T cells (Treg), Th17 and T folli-cular helper (Tfh) cells (Zhou et al., 2009; O’Shea andPaul, 2010). Th1 cells produce IFN-g, TNF-a and IL-2important for activating intracellular microbicidal activitiesand for the generation of CD8+ cytolytic T lymphocytes(CTL), all of which are required for control of intracellularpathogens. Th2 cells produce IL-4, IL-5 and IL-13 whichenhance antibody responses and antibody-dependentcellular responses required for control of extracellularpathogens. Th3/Treg cells produce TGF-b, IL-10 and/orIL-35, and are associated with potent secretory IgAresponses protective against mucosally invasive patho-gens and minimization of immunopathology duringchronic infections. Th17 cells produce IL-17, IL-21 andother cytokines/chemokines that enhance inflammatoryrecruitment of neutrophils and additional T cells, macroph-ages and dendritic cells. Th17 cells may be important forboth direct control of extracellular pathogens along epi-thelial surfaces and indirect enhancement of protectionagainst intracellular pathogens by recruitment of Th1 cells(Khader et al., 2007). Tfh produce IL-21 in germinalcentres important for B-cell activation, differentiation andaffinity maturation.

Depending on the nature of the target pathogen, avaccine may need to induce Th1 responses to protectagainst an intracellular pathogen, Th2 responses toprotect against an extracellular pathogen, or Th17 andTh3 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 cytokinestrigger 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 biology3. Need for CD8+ T cells For intracellular pathogens, especially if replicates in cytoplasm and/or infects

non-haematopoietic cells4. 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 memory6. Adjuvant selection Alum salts enhance Ab responses; Toll-like receptor agonists, oil/water emulsions enhance

T cells7. Vectors/delivery format B- versus T-cell responses, ideally mimic natural pathogen invasion strategy8. Schedules and routes of vaccination Mucosal versus systemic vaccinations depending on pathogen biology, heterologous

boosters9. Immunological networks/biomarkers Identify molecular signatures associated with optimal immune response, mucosal versus

cutaneous trafficking10. 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

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 moststable end-point phenotypes among these subsets, andhave been shown to be programmable in long-termmemory cells (Swain, 1994). In contrast, Th17, Th3/Tregand 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 abovementioned differential driving cytokines, and other factorsaffecting the distinct transcriptional activation associatedwith these subsets, can be used to selectively induce themost relevant immune responses for the target pathogen(Hoft and Eickhoff, 2002).

Additional subsets of T cells may be important targetsfor new vaccines, broadening the recognition potential ofthe immune response and/or facilitating the rapid recall ofimmunity. Certain T cells can recognize ceramides andother lipids presented by non-polymorphic CD1 mol-ecules, and can rapidly produce cytokine responses uponrestimulation (Hiromatsu et al., 2002; Vincent et al.,2003). Similarly, the g9d2 TCR+ human T-cell subset canrecognize phospholipids in an MHC-unrestricted fashion,can rapidly produce cytokines capable of enhancing Th1immunity and can directly inhibit intracellular pathogenreplication (Hoft et al., 1998; Shen et al., 2002; Moritaet al., 2007; Spencer et al., 2008). Inclusion of key cera-mide and/or lipid antigens for specific activation of NKTand/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 peptideepitopes presented by MHC class II molecules whichgenerally sample extracellular antigens taken up bypinocytosis/phagocytosis, or antigens synthesized bypathogens that persist and/or replicate within the endoso-mal compartments of infected cells. CD8+ T cells arestimulated by foreign peptides presented by MHC class Imolecules which sample cytoplasmic contents of infectedcells. These CD8+ T cells contain cytolytic granules withperforin and granzymes responsible for membranedamage and apoptosis induction in infected target cells.Another molecular component of human cytolytic granulesis granulysin which can mediate direct microbicidal activityagainst bacterial pathogens (Stenger et al., 1998). In addi-tion to this classical effector degranulation response withsubsequent lysis and death of the recognized target cell,CD8+ T cells can trigger the inflammasome and/or lead tonon-lytic effects that enhance intracellular suppression ofcertain pathogens like HSV (Knickelbein et al., 2008;Metkar et al., 2008). Because CD8+ T cells are generallystimulated by cytoplasmic peptides, vaccines that result in

synthesis of antigens within the antigen presenting cell(e.g. live attenuated vaccine vectors and DNA expressionvectors) are needed to induce optimal vaccine-specificresponses. However, cross-presentation of soluble extra-cellular antigens by dendritic cells and B cells to CD8+ Tcells can occur with the right adjuvant conditions (e.g. TLRsignals 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 beenidentified with complementary roles (Sallusto et al., 1999;2004; Willinger et al., 2005). CCR7-expressing centralmemory T cells (Tcm) with high proliferative potentialre-express lymph node homing receptors which allow foraccumulation in lymph nodes. CCR7 negative, peripheraleffector memory T cells (Tpem) are distributed throughoutperipheral tissues, have little proliferative potential, butare able to rapidly provide protective effector functions atthe initial peripheral sites of pathogen rechallenge. Tcmserve as efficient immune memory storage facilitieswaiting for dendritic cells from the periphery to bring earlyantigens upon remote pathogen rechallenge to the drain-ing lymph nodes where Tcm undergo restimulation, pro-liferative expansion and effector differentiation to dampenlater waves of pathogen replication and spread. Tpemprovide a first line of adaptive immune defence and gen-erally require persistence of antigen for their prolongedpresence. 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 provideoptimal vaccine efficacy. However, experimental vaccinesdesigned to maintain long-term induction of Tpem withchronically persisting vaccine vectors, with the goal ofinducing optimal protection against initial epithelial inva-sion immediately upon pathogen exposure are beinginvestigated (Hansen et al., 2009).

In addition to proliferative expansion and effector T-cellfunction, the polyfunctionality of vaccine-induced memoryimmune responses is another important feature for con-sideration in vaccine development. For example, severalstudies have found that antigen-specific IFN-g productionis not the only effector function important for optimal pro-tective immunity. Seder et al. demonstrated that thecapacity of CD4+ T cells to produce all three Th1 cytok-ines, IFN-g, TNF-a and IL-2, was a much better predictorof the relative protective capacity provided by variousleishmania vaccines in murine models than IFN-g produc-tion alone (Darrah et al., 2007). Similarly, CD8+ T cellscapable of further proliferative expansion and productionof both IFN-g and perforin were associated with delayedHIV disease progression while CD8+ T cells capable ofproducing only IFN-g were not (Migueles et al., 2002).

936 D. F. Hoft, V. Brusic and I. G. Sakala


Consistent with these other results, we have shown thatIFN-g production alone does not correlate with the abilityof human T cells to inhibit intracellular mycobacteria (Hoftet al., 2002). Therefore, polyfunctional assessments ofthe memory T cells induced by vaccination are importantfor clinical development of the most effective vaccines.

Topic 5: Treg

Regulatory T cells are important for negative regulation ofimmunity, and the prevention of autoimmune diseases(Bluestone and Abbas, 2003). Natural and induced Tregdevelop in the thymus versus periphery, respectively, andcan limit over-exuberant immune-mediated inflammation.Natural Treg express Foxp3, a master transcription factorthat induces a detailed genome-wide molecule pro-gramme that maintains the Treg phenotype, and suppresseffector T-cell responses through contact-dependent and-independent mechanisms. Induced Treg may or notexpress Foxp3, but still can inhibit effector T-cellresponses through the production of IL-10 and/or TGF-b.Treg in general are critical for maintaining homeostasis ofthe immune system and minimizing the pathologicaleffects of immune activation. However, many infectiouspathogens induce Treg which can interfere with the devel-opment of optimal protective immunity. In fact, Belkaidet al. demonstrated that Treg can be responsible for thepersistence 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 beenhanced by limiting or actively inhibiting Treg responses.Furthermore, there is some evidence that certain T-cellepitopes can at least preferentially induce Treg ratherthan effector T-cell responses (Massa et al., 2007). Avoid-ing Treg biasing epitopes obviously could be important forthe development of some types of vaccines.

In addition to the natural and induced Treg discussedabove, natural ligands produced in vivo during comple-ment activation can trigger signalling via CD46 on humanT cells leading to negative feedback induction of IL-10production in Th1 cells after peak immune responses arefinished and no longer needed (Kemper et al., 2003;Cardone et al., 2010). Several pathogens have evolved toutilize CD46 as a specific target receptor for infection ofhost cells, and engagement between these pathogensand CD46 can trigger negative feedback inhibition of cell-mediated immune responses (Kemper and Atkinson,2007). These negative regulatory effects triggered duringinitial infection can prevent the induction of optimalvaccine-induced immunity. We have shown that BCGimmunity in humans is reduced by the effects of CD46cross-linking on T cells, and natural ligands produced

during BCG infection/replication can specifically enhanceCD46 signalling (Truscott et al., 2010).

Learning how to prevent these downregulatory signal-ling events may be important for learning how to inducemore protective vaccine-induced immune memory. On theother hand, IL-10 produced by Treg has been implicatedas 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 optimizefuture vaccine strategies.

Topic 6: Adjuvants

Specific adjuvants can enhance immune memory andshape the phenotype of the recall response. Until veryrecently, the only adjuvants approved for human use werealuminium salts, which can increase the half-life of anantigen, improve uptake by professional phagocytes andtrigger the inflammasome via NALP3 sensing (Eisenbarthet al., 2008). Alum adjuvants clearly increase the titres ofspecific antibodies generated by vaccination, but are notoptimal adjuvants for the induction of CD4+ Th1 cell andCTL responses important for the control of intracellularpathogens. Newer adjuvants including oil/water mixtureswith toll-like receptor triggering ligands (e.g. lipid A forTLR4, unmethylated CpG dinucleotide motifs for TLR9,etc.) are becoming available which can greatly enhanceTh1 and CTL responses (Coffman et al., 2010).

Classic immunological work has confirmed that Th1 andTh2 priming can induce stable differentiated phenotypesthat maintain their original bias after remote recall stimu-lations (Swain, 1994). Systemic IL-12 administration (oradministration of a TLR ligand such as a CpG oligonucle-otide that stimulates IL-12 production by dendritic cellsand macrophages) during vaccination in animals canclearly bias for Th1/CTL response generation. SystemicIL-4 given during vaccination can skew for long-term Th2memory immune cells. It is not clear yet whether Th3,Treg, Th17, NKT and/or gd T cells can develop similarstable long-term differentiated effector memory cells.However, driving cytokines have been identified for atleast short-term induction of these latter immune subsets(e.g. TGF-b/IL-10 for Th3/iTreg generation; TGF-b plusIL-6, IL-21 and IL-23 for Th17 generation) (Zhu et al.,2010) and future research will focus on the importance ofthese additional subsets and their potential for attaininglong-term stable immune memory phenotypes. Additionalresearch will need to explore the relative values of usingdifferent membrane and cytoplasmic foreign sensors(TLR, RLR and NLR) individually and in combinations toinduce the best vaccine-specific responses appropriatefor each unique pathogen.



Topic 7: Vectors/delivery format

Next for consideration are a diverse group of deliverysystems all designed to result in antigens being synthe-sized within or translocated into the cytoplasm of antigenpresenting cells in order to enhance T-cell stimulation andfacilitate class I presentation to CD8+ CTL. Many viral andbacterial live attenuated vectors have been developedand shown to induce potent cell mediated immunity inanimal models. These live attenuated vectors are highlyimmunogenic usually producing multiple pathogen-associated molecular patterns that signal through TLR/RLR/NLR, providing the inflammatory signals andco-stimulation required to optimally induce protectiveimmune responses. In addition, these live attenuatedvectors can be delivered through more natural routes ofpathogen invasion (e.g. through mucosal surfaces) poten-tially inducing more relevant regional immune responsesfor protection against the initial infection. However, byinducing potent inflammation these highly immunogeniclive vectors can be associated with undesired reactoge-nicity, or may induce vector-specific immunity that canreduce the efficacy of booster vaccinations with the samevaccine.

DNA vaccines, consisting of recombinant plasmidsencoding vaccine antigens under the control of a eukary-otic promoter and containing TLR9 stimulatory CpGmotifs, also deliver genes for expression inside the cyto-plasm of antigen presenting cells. These plasmid vaccinevectors have induced very potent Th1 and CTL responsesin animal models but have not been as successful inhumans to date. However, methods involving electropo-ration of plasmid into the superficial epidermis with anapplied electrical current recently have been developedwhich result in enhanced targeting and uptake by residentLangerhan and other dendritic cells, and are likely toimprove human DNA vaccinations (Hirao et al., 2011; Linet 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 tendto be taken up by mucosal surfaces and concentratewithin highly phagocytic cells (Heurtault et al., 2010;Mishra et al., 2010). This is a very active area of researchcurrently, although so far there are no licensed vaccinesconsisting 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 theappropriate 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 vaccinemust be optimized. For pathogens that invade through orare shed from mucosal tissues, mucosal vaccinationsmight induce optimal protection against initial infectionand/or secondary transmission. Activation of T and B cellswithin immune inductive sites lining mucosal tissues leadsto the upregulation of surface molecules on these cellsimportant for allowing access to peripheral mucosal sitesafter circulation in blood. In fact, a network of mucosalimmunity known as the common mucosal immune systemresults from the expression of mucosal homing moleculeson immune cells activated in one mucosal tissue that canlead to enhanced distribution of these immune cells alongmost if not all mucosal tissues. Specific integrin com-plexes, additional adhesion molecules and chemokinereceptors are involved in the specific dissemination ofmemory immune T and B cells to mucosa (Kunkel andButcher, 2002). For example, the a4b7 integrin complex isupregulated on the surface of lymphocytes activated inthe Peyer’s patches. This integrin specifically binds toMadCAM1 on endothelial cells and triggers transendothe-lial migration from the vasculature into the peripheralmucosal tissues. In contrast, intradermal or subcutaneousvaccinations induce cutaneous lymphocyte antigen (CLA)expression on T and B cells activated in the skin or lymphnodes receiving lymphatic drainage from cutaneous sites.CLA is important for recognition of molecular structureslining endothelial cells in cutaneous microcapillariesrequired for transpedesis into the peripheral cutaneoustissues. Therefore, by changing the route of a vaccination,regional immune responses relevant for protectionagainst specific pathogens can be selectively enhanced.

The optimal number of booster vaccinations must bedetermined for each new vaccine. Generally, more than 1dose of a vaccine is required to induce optimal immuneresponses. Booster vaccinations lead to higher titre anti-body responses with increased affinity resulting fromsomatic hypermutation induced by recurrent stimulationwithin the hypervariable regions critical for antigen recog-nition. Two- to four-week intervals between booster vac-cinations historically have worked well for vaccinesdesigned to induce protective antibody responses. Muchless is known regarding the optimal boosting intervals forinduction of Th1, CTL and other specific T-cell subsets.Although somatic hypermutation is thought not to occur inantigen-specific T cells, the most relevant clones areselectively amplified in response to booster vaccinationsbased on affinity reactions between MHC/peptide andTCR. These higher avidity T cells can lead to enhancednumbers of antigen-specific T cells, potency of effectorresponses and longevity of antigen-specific memory.Whether boosting T-cell responses after completion oftheir differentiation into maximal numbers of resting Tcm



could improve long-term T-cell immune memory isanother important hypothesis to test.

As mentioned above, vectors can induce vector-specific immunity that can limit the effectiveness ofbooster vaccinations with the same vaccine vector.Another important concept to investigate during vaccineschedule optimization is whether distinct molecularvaccine formats presenting the same vaccine antigens inheterologous prime/boosting combinations can enhancevaccine-induced protective memory immunity (Schneideret al., 1998; Wei et al., 2010). Priming with a DNA vaccinefollowed by boosting with a viral vectored vaccine, whenboth vaccines encode the same pathogen-specific anti-gens, can greatly enhance at least the total numbers ofantigen-specific T cells that develop and persist long term.This technique of using heterologous prime/boostingschedules can overcome the problems encountered dueto vector-specific immunity that develops during homolo-gous prime/boosting vaccinations, and may maximize thebreadth and phenotype of immune subsets induced byproviding 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 weapproach vaccinology (Pulendran et al., 2010). Genome-wide expression comparisons of effector T cells andresting Tcm have demonstrated that the most potenteffector cells represent the most terminally differentiatedcells with the least capacity for self-renewal (Willingeret al., 2005). In contrast, Tcm are less terminally differen-tiated, have the best self-renewal/expansion capacity andbecause of increased expression of anti-apoptotic genesprovide more long-term populations of antigen-specificmemory T cells.

Genome-wide expression pattern analyses have impli-cated basic metabolic pathways in effector versusmemory T-cell generation. The mTOR pathway, activatedby the presence of numerous substrates and important forgrowth and activation of primarily activated T cells, canbias for development of mostly short-lived effector T cells(Araki et al., 2009). Specific inhibitors of the mTORpathway (e.g. rapamycin and metformin) can lead toincreases in long-term memory T cells present after vac-cination. Further network analyses of immune cells haveidentified vitamin D metabolism as important for maximiz-ing the cutaneous trafficking potential of memory T and Bcells, while vitamin A metabolism is involved in the induc-tion of mucosal trafficking potential (Sigmundsdottir andButcher, 2008). The near future should bring additionalgenome-wide expression studies that identify gene net-

works induced early on after vaccination that can predictoptimal long-term protective immunity and characterizethe more detailed factors involved in differential develop-ment of Th1/Th2/Th3/Treg/Th17 immune phenotypes anddistinct lymphocyte homing programmes.

To demonstrate the power of molecular transcriptomalanalyses, we include preliminary data that we haverecently generated studying human volunteers with previ-ous exposure to mycobacterial antigens. We have con-ducted a series of tuberculosis (TB) vaccine trials with theoverall goal of learning how to improve the protectivecapacity of new TB vaccines. To our knowledge, there areno published reports of human T-cell antigen-specificmolecular transcriptomes. Transcriptomes expressed insubsets of unactivated, total polyclonal human memoryT-cell subsets have been studied, but not antigen-specificpopulations or the differences between rested and acti-vated memory T-cell responses. In mice, TCR transgenicmodels have facilitated studies of antigen-specific tran-scriptional profiles by providing highly purified populationsof 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 arerelated to the antigen-specific populations of interest. Inhumans, the frequencies of T cells specific for a givenvaccine or pathogen usually are < 1–10% of the totalT-cell population. Thus, it has been assumed that antigen-specific T cells must be purified in order to study complexgene 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 smallnumber of T cells recovered, the consequent need foramplification techniques to study the mRNA expressed,and potential alteration in gene expression profiles due tolabour intensive in vitro manipulations. To address thesefeasibility concerns, we completed a pilot experimentwhich clearly demonstrates that TB-specific transcrip-tional profiles can be studied with a relatively simpleapproach not requiring purification of antigen-specific Tcells. 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-foldincreases in IL-2 mRNA at 24 h in T cells stimulated withBCG-infected compared with uninfected DC. Theseresults demonstrate that we can detect marked increasesin expression of the IL-2 target gene despite the fact thatBCG-specific T cells likely represent only a minor fractionof all polyclonal CD4+ memory T cells, and indicate thatstimulation of T cells for 24 h with BCG-infected DC(moi = 20) represent reasonable conditions for genomeexpression studies of BCG-specific T-cell responses. Weprepared cDNA and cRNA from RNA samples harvestedfrom CD4+ memory T cells co-cultured with uninfected



and BCG-infected DC for 24 and 48 h from these threevolunteers and performed Affymetrix hybridizations withHG-U133 Plus 2 Affymetrix chips. The data were normal-ized and ANOVA used to identify genes with significantchanges in expression comparing BCG-infected and unin-fected stimulation conditions. We found 3098 genessignificantly altered by stimulation with BCG-infectedDC and 608 upregulated at both the 24 and 48 h timepoints. Gene set enrichment analysis (GSEA) looking fornetworks of genes altered by BCG antigen-specific stimu-lation indicates that many gene sets classically associ-ated with immune response pathways (T-cell receptorsignalling, Jak-Stat signalling, apoptosis, cytotoxicity,cytokine–receptor interactions and integrin-medicated celladhesion) are highly represented among the significantlyaltered gene expression patterns (http://cvc.dfci.harvard.edu/share_folder/IMN). Therefore, we can use a relativelysimple strategy to purify total memory CD4+ T cells andstudy the expression of a highly diverse set of humangenes induced by antigen-specific stimulation. We arecurrently using this strategy to identify specific alterationsin the antigen-specific gene set associated with mucosalversus cutaneous BCG vaccination, and which genes

predict the best functional long-term memory responsesafter BCG vaccination. The overall goal is to determinenew biomarkers that can be used to more rapidly andaccurately assess mucosal and systemic immunogenicityof iterative TB vaccination approaches.

Topic 10: Clinical development/safety andsurrogate markers

Once a vaccine is ready for clinical testing, the focus firstbecomes safety. Phase I dose escalation trials aredesigned to identify the safest and most immunogenicvaccine doses. Phase II trials expand safety analyses intolarger numbers of volunteers and begin to address theefficacy of vaccination. Phase III trials are designed toprovide sufficient statistical power to definitively addressvaccine efficacy and include detailed reactogenicityassessments. Throughout the clinical developmentpathway from phase I to phase III, it is important to havesurrogate markers of protective immunity that can beassessed in vaccinated volunteers. Ideally, correlates ofprotection should be known to help direct vaccine optimi-zation. Unfortunately, correlates of protection are not

Fig. 1. Human molecular transcriptomalanalyses can help identify important biologicalresponses required for successful vaccines. In(A), memory CD4+ T cells were purified fromthree PPD+ persons with Miltenyi negativeselection kits resulting in > 97% pure memoryCD4+ T cells. Memory CD4+ T cells werestimulated with autologous DC (20:1 T : DCratio) that were uninfected or infected with aBCG moi of 4, 20 or 100. Total RNA washarvested at 24, 48 and 72 h. We completedqRT-PCR for IL-2 mRNA to determine whatconditions gave us the best ability to seeBCG-induced changes. RNA harvested fromthe optimal conditions was used to identify theAffymetrix molecular signatures associatedwith BCG-specific stimulation demonstratingthat the expression of 3098 genes wassignificantly involved (data not shown). (B)depicts the major phases of T-cell activationand the serial points of transcriptomalanalyses being used to identify: (i) geneexpression patterns involved in programminglong-term protective immune memory, and (ii)the differential gene expression patterns thatpredict T-cell responses capable of providingoptimal mucosal versus systemic immunity.



known for many of the human pathogens that remain keychallenges for vaccine development. For HIV, TB andmany other major world pathogens, only partial informa-tion regarding surrogates/correlates of protective immu-nity is available. This shortcoming makes iterativeresearch critical involving the empirical development ofvaccine candidates, clinical development of experimentalvaccines and refinement of second-generation vaccinesbased on enhanced targeting of new surrogates/correlates identified in vaccine trials with prototype vac-cines. Finally, once a vaccine is shown to be safe andefficacious in humans, additional research is necessary toassess ongoing effectiveness of the vaccine for preven-tion of infection and disease due to the target pathogenunder real-world conditions, and to provide quality controlfor continual production of effective vaccines.

Concluding remarks

Vaccine development is a complex process involving mul-tiple different specialists, careful thought into the specificvaccine design, as well as laborious testing and evalua-tion. We have discussed some of the key issues importantfor the generation of a successful vaccine. Because ofspace limitations we have left out many additional stepsthat are necessary for this process including detailedanimal testing of immunogenicity, protective capacity andtoxicity. We hope that the reader now has a more com-plete appreciation of the detailed requirements for devel-opment of a successful vaccine.

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