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Vaccine 30 (2012) 4927– 4933

Contents lists available at SciVerse ScienceDirect

Vaccine

jou rn al h om epa ge: www.elsev ier .com/ locate /vacc ine

eview

ew technologies for new influenza vaccines

lan Shaw ∗

axInnate, 3 Cedarbrook Drive, Cranbury, NJ 08512, United States

r t i c l e i n f o

rticle history:eceived 28 January 2012eceived in revised form 24 April 2012ccepted 26 April 2012vailable online 9 May 2012

eywords:

a b s t r a c t

The currently available influenza vaccines were developed in the 1930s through the 1960s using tech-nologies that were state-of-the art for the times. Decades of advancement in virology and immunologyhave provided the tools for making better vaccines against influenza. We now have the means to makevaccines that address some of the shortcomings of the original products, in particular performance in theelderly.

© 2012 Elsevier Ltd. All rights reserved.

nfluenzaaccineirus-like particlesttenuatedold-adaptedS1 deletion

oll-Like Receptors

Influenza vaccines have been a feature of the routine immuniza-ion portfolio since the 1960s. These vaccines, developed with theest technology available at the time, have served us well. Overhe past several years there has been a growing appreciation of thehortcomings of the original egg based vaccines’ immunogenicity,specially in the elderly, where 40 years of clinical studies haveailed to come up with a satisfactory solution. A recent review ofhis topic from CIDRAP highlights the need to develop a new gen-ration of influenza vaccines that take advantage of the last 60ears of advancement in immunology and molecular biology [1].hree major events have driven a renewed interest in influenzaaccines. In the late 1990s there were widely publicized outbreaksf avian influenza in Asian bird markets that led to lethal infec-ions in humans. Fortunately, human-to-human transmission ofvian H5 virus is very rare, if it happens at all. The specter of suchransmission, if it became efficient, was the impetus for a wave ofew interest and investment in flu vaccine technology. The secondajor event was a realization over time that the standard trivalent

nfluenza (TIV) vaccines are relatively poor immunogens in peo-le over 65 years of age; this is the fastest growing segment of ouropulation. Beginning in the 1960s, a series of clinical studies of TIVere carried out in an effort to find a more immunogenic dose and

egimen. Most of these studies focused on increasing the dose fromhe standard 15 �g per component up to as much as 405 �g of HA,ith the best results showing a doubling of the HAI titer. Recently,

∗ Tel.: +1 215 208 9036.E-mail address: alan.shaw@vaxinnate.com

264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.vaccine.2012.04.095

a “4×” dose vaccine with 60 �g of each component was approvedfor people over 65. The third event was the pandemic of 2009. Weexpected an avian virus pandemic, and we got a swine-human virusinstead. The influenza manufacturing community applied the bestpossible effort, but the vaccine became available far too late andwent largely to waste. Together, these three elements now drive anew interest in influenza vaccines.

This article will provide a very brief history of the developmentof the original egg-based vaccines and the technical refinementsthat have been applied to them. We will also cover the evolution oflive nasally delivered vaccines and their commercial development.

We will devote the majority of our attention to the avalanche ofnew approaches triggered by a renewed interest in flu following theavian influenza outbreaks, our experience with the 2009 pandemic,and the impact of funding for influenza vaccine development pro-vided by the US government. Since this is now a fast-moving field,much of the core data, especially clinical results, are not publishedin a final form that is suitable for citation. Where appropriate, sum-maries of public presentations will be included. The goal here is toprovide an overview of where the influenza vaccine field is headedand why rather than to compile a complete list of projects.

1. A short history

Influenza virus was first isolated in the early 1930s by British,

American and Australian investigators working at the forefront ofwhat we now know as clinical and veterinary virology. The viruswas detected initially due to its ability to cause disease in ferrets.Ironically, the use of the ferret for influenza work is at least partly

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oincidence, as the UK lab’s initial mission was to work on canineistemper virus which was affecting a substantial fraction of dogs

n the UK at the time.

. Egg-based vaccines

In relatively short order, this group worked out the culturef influenza virus in minced chick embryo, in eggs, and then inhe allantoic cavity of eggs. Shortly thereafter in the early 1940s,emagglutination was recognized both as a means of titrating viruss well as antibody against the virus. The single radial hemolysisssay [2] was developed in the mid-70s in response to a need for

better means of dosing HA. These basic tools continue to sup-ort the production and analysis of influenza vaccines to this day.

good account of these events can be read in David Tyrell’s Chapter in The Textbook of Influenza edited by Nicholson, Webster and HayBlackwell).

The first useful influenza vaccines can be traced to formalin-nactivated virus preparations made in mouse lungs or chickmbryos from 1937 through the early 1940s. The US Army carriedut a series of studies that demonstrated protective efficacy in mil-tary personnel beginning in 1942 using formalin-inactivated virusrown in eggs and concentrated by centrifugation. The first vaccineicensed by the “FDA” of the time was made by Parke-Davis, and theecond was made by Merck.

In the 1960s, zonal centrifugation was implemented as a betterethod for enrichment of the virus-containing fraction of allan-

oic fluid, and “split” virus vaccines were introduced. Treatmentf the enriched virus with either ether or deoxycholate disruptedhe virion with the effect of reducing adverse reactions associatedith the earlier whole virion preparations. Whole inactivated virus

accine, at a lower dose, has been reintroduced recently by Bax-er. The next advance in the late 1960s was the implementationf “high growth reassortants” [3] where the HA and NA genes areelectively crossed into the PR8 strain that grows well in eggs. Thisrovided a more reliable means of propagating influenza virus atcale and greatly improved the match of the vaccine. At this point,he inactivated “flu vaccine” became a routine product made bytandard methods by numerous commercial vaccine producers.ecently, reverse genetics has become the preferred method foraking production strains.

. Cold adapted live attenuated vaccines

In parallel, beginning in the early [4] 1960s Smorodintsev’saboratory in Leningrad [5,6] began the development of live andive-attenuated vaccines based on a cold-adapted and temperatureensitive virus derived from the 1957 Leningrad isolate. At the Uni-ersity of Michigan, Maassab initiated a similar program based onhe Ann Arbor A and B strains [7]. The Ann Arbor-based vaccine,ow known as FluMist®, was licensed for use in the US in 20038]. The Leningrad-based vaccine is widely used in Russia and isvailable for license via the WHO.

Vaccines are public health tools, but they are also manufacturedroducts made, for the most part, by companies that have to make

profit. This mix of business, public health and public policy in highly regulated environment makes an exciting and rewardingxistence for those engaged. However, the path from concept toicensed vaccine is not always linear.

Two good examples of tortuous business pathways can be seenith the live attenuated vaccines. The live-attenuated, cold adapted

asally delivered vaccines from Leningrad and Michigan took over0 years of development. The Michigan/Maasaab vaccine began as aniversity-NIH project, which was licensed to Wyeth and returnedwice, then licensed to Aviron, a California Biotech Company who

012) 4927– 4933

licensed the vaccine to Wyeth (again) in 1998–1999. Wyeth soldthe project to MedImmune (who acquired Aviron as part of thedeal) where development was completed. FluMist® was approvedfor marketing in the US in 2003. The Leningrad/57 vaccine also hasa complicated commercial history. Development began at the Insti-tute for Experimental Medicine around 1960 with numerous smallclinical studies over decades and approval for use in Russia. In 2002Merck licensed the Leningrad/57 strains and spent three years char-acterizing the genetics of the vaccine and developing processes formanufacturing [9–11]. Ultimately, Merck returned the rights to theLeningrad/57 strains to the Institute. Subsequently, the Leningradstrains were licensed to Nobilon, a division of Schering-Plough. In2010, Merck merged with Schering-Plough, and as a consequence,the Leningrad strains are now back at Merck! The point here is thathaving a developed vaccine is not sufficient for success. A consis-tent business plan is also a necessary part of the equation. This isan element of all of the new vaccine programs on the horizon.

4. New initiatives in influenza: expanding capacity

The standard trivalent inactivated influenza vaccines (TIV)became routine products made by standard methods from the1960s to the present day. US sales were in the range of about 20million doses per year up to the 1990s when a focus on influenzamorbidity and mortality raised the general awareness. The out-breaks of influenza in the poultry markets of Asia in 1997–1998spurred an effort by the major flu vaccine producers to explore thepossibility of expanding their capacity beyond the current marketdemand under the anti-trust provisions of the IFPMA in Geneva.This was not a simple task, and it was a business risk and bur-den. The vaccine producers made the argument that in order tomaintain the capacity to deal with a pandemic, there should be abetter uptake of seasonal vaccine. However, having a significantover-capacity can lead to lower prices that in turn cause produc-ers to exit the business. This is what happened in the US duringthe 1970s and 1980s and led to the US having a single domestic fluvaccine source, sanofi-pasteur. Having excess idle capacity is notgood business. At a recent WHO Influenza vaccine supply meeting(12–14 July, 2011) the International Federation of PharmaceuticalManufacturers Association estimated that current credible demandfor TIV is 553 million doses per year, while annual capacity is 1383million doses. The limit is currently not volume, but in the faceof a pandemic as in 2009, the crucial element is time to last dosedelivered.

Expanding capacity has provided an opportunity to increase thenumber of virus types included in the seasonal vaccine. The cur-rent vaccines comprise an H1, an H3 and a single B-strain. TheB-strains are typically difficult to grow, consuming a disproportion-ate fraction of the manufacturing effort. Expanded capacity allowsthe inclusion of a second B-strain, something that has been dis-cussed for years but has not been implemented. Several tetravalentvaccine development projects are now underway.

5. Year-round egg supply

Up until around 2001, the chicken flocks supporting vaccine pro-duction were expanded to meet seasonal vaccine manufacturingfrom February to August, and were then culled, only to be expandedagain for the next season. The first “novelty” for the flu vaccine busi-ness was an agreement in 2002 by the Secretary of US Health andHuman Services (USHHS) to fund the maintenance of chicken flocks

year-round so as to facilitate emergency ramp-up of vaccine pro-duction. Note that a hen must be at least 20 weeks old before layingeggs. This arrangement for year-round flock maintenance remainsin force and will help with the timing of initiation of manufacturing.

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. Mammalian cell culture

The next advance, also funded in part by USHHS, was the imple-entation in the US of mammalian cell culture-based influenza

accine production. Cell culture is attractive as it does not relyn the maintenance of huge flocks of chickens, and it may belightly faster. Contracts were awarded to three producers (sanofi-asteur, Novartis and Solvay) for US-based mammalian cell culture

nfluenza vaccine production facilities and development. Ulti-ately, Solvay and sanofi-pasteur terminated their programs for

nancial and feasibility reasons. The Novartis facility in North Car-lina is now complete and ready to begin production. The mainemaining hurdle is process economics. Novartis has a cell-cultureased vaccine licensed in the EU, as do Baxter and Solvay.

. Adjuvanted vaccines

Influenza vaccines comprising an adjuvant have been availablen Europe for several years. The primary target population for theseaccines has been those over 65 years of age, in whom the immuneesponse is less robust and who suffer greater morbidity and mor-ality from influenza. During the course of the 2009 pandemic,djuvanted influenza vaccines were used widely in Canada and inurope, but not in the US. We anticipate that these adjuvanted vac-ines will be approved in the US at some point. Addition of oil-waterased emulsion adjuvants to standard egg-based vaccines allows a-fold dose sparing effect. At a recent 2012 conference, Novartishowed that Fluad, their MF59-adjuvanted vaccine provided twicehe efficacy of standard TIV in children aged 6–72 months.

. Recombinant DNA based vaccines made in insect cells

One of the biggest variables in influenza vaccine productions the ability of a given seasonal virus production strain to grown eggs. This was one of the main reasons that the 2009 pan-emic vaccine was delayed. Production strains grew poorly andequired sub-selection and refinement that took precious time.odern rDNA methods can circumvent the need to propagate the

irus. The major immunological target on the virus is hemagglu-inin (HA) which carries the vast majority of neutralizing epitopes.

variety of methods for making influenza HA have been developednd tested in the clinic. In order of chronological development:

Protein Sciences produces influenza hemagglutinin, FluBlok, innsect cells (Army Worm, Spodoptera frugiperda) infected with annsect cell virus vector (Baculovirus). This is now a well-accepted

ethod for making vaccines; one of the licensed human papillo-avirus vaccines, Cervarix® is made using this general system.

he Protein Sciences vaccine comprises the whole HA sequence,ielding a mix of HA monomers and HA rosettes connected via therans-membrane sequence [12–14]. Clinical trials have shown thisaccine to be well tolerated and immunogenic at a dose of 45 �gor each of the three TIV components [14–16]. FluBlok has beenubmitted to the USFDA for approval. Early in the development ofhis vaccine, a candidate pandemic vaccine was made based on theong Kong156/97 strain. This vaccine, like many H5 vaccines thatame afterwards, was suitably immunogenic only after two dosesf 90 �g [17].

Novavax produces virus-like particles in insect cells infectedith a baculovirus vector, analogous to the process used by Protein

ciences, above. The virus-like particles contain hemagglutinin,euraminidase, and the influenza matrix protein. Inclusion of the

atrix protein allows HA and neuraminidase to associate withatrix so as to form a particle that resembles an influenza virion

18]. The advantage of the particulate presentation is that den-ritic cells have a particle-recognizing mechanism that takes up

012) 4927– 4933 4929

virus-sized particles [19]. This in turn activates the dendritic cell.The currently licensed hepatitis B and human papillomavirus vac-cines rely on this type of particulate presentation. These influenzavaccines are immunogenic across doses of 5–45 �g per HA species[20], and they elicit cross-clade protection in animals and inhumans vaccinated with H5 avian HA-containing VLPs [21,22].Addition of a liposomal adjuvant may improve immunogenicity[23].

9. Recombinant DNA based vaccines made in plants

Modern molecular biology provides the ability to expressrecombinant proteins in a variety of cell types, including plant cells.Two groups have successfully made HA in tobacco plants.

Fraunhofer Institute [24] uses a tobacco mosaic virus vector toexpress HA in tobacco plants. The virus vector DNA in solutionis instilled into the plants by inverting them and submerging theleaves. A vacuum is applied, opening the stoma on the bottom ofthe leaves, and drawing the DNA inside the plant. HA is expressed,and the antigen is extracted from the leaves. This vaccine has beenshown to be immunogenic in a phase 1 clinical study VIDADI.

Medicago [25] uses a similar approach in tobacco plants,using an Agrobacter vector introduced by vacuum instillation. Theexpressed HA sequence in the Medicago system creates a virus-like particle. Doses of an H5 Indonesia vaccine ranging from 0.7 to11 �g of VLPs absorbed onto alhydrogel elicited robust HAI titersin a clinical trial [26].

Yields and purity of both plant-based influenza vaccines are con-sistent with the requirements of an industrial process that shouldsatisfy regulatory agencies.

While the major focus on plants has been centered on tobacco,there are many other options. Enough is now known about thegenetics and regulation of gene expression in organisms other thanmammals, yeast and bacteria, to expand the repertoire of hostsfor flu vaccine production. Two examples presented at a recent“New Cells for New Vaccines” meeting included the common but-ton mushroom and brine shrimp.

10. HA-based influenza vaccines made in bacteria

Since the early days of recombinant DNA influenza has been avaccine target of interest. Numerous groups have taken up the chal-lenge. One of the pioneers in this area is the Arnon lab in Israel[27–30]. Since the hemagglutinin molecule is a complex foldedstructure, the Arnon lab created as series of peptide epitope basedvaccines comprising conserved structures of HA as well as nucleo-protein. These vaccines, when adjuvanted, perform well in animalmodels. This effort, which continues aggressively at BiondVax, hasbeen the subject of a recent review [31].

For decades, the influenza vaccine dogma held that the hemag-glutinin molecule had to be produced in a vertebrate cell substratethat could fold, glycosylate and secrete the protein. It turns outthat none of this is true. First, a group at the CDC showed, usinga DNA vaccine, that knocking out the various N-linked glycosyla-tion sites on HA had no measureable impact on immunogenicity inmice [32]. Subsequently, two groups have produced influenza HAin Escherichia coli. At the USFDA/CBER, Hana Golding and her teamhave produced a properly-folded H5 VietNam HA molecule that isimmunogenic in animals. In addition, this group has identified apotential new oligomerization sequence that may be useful [33].

VaxInnate has developed a method for making HA-based

influenza vaccines based on a genetic fusion of the sequence for theHA globular head with the sequence for flagellin, the ligand for Toll-Like Receptor 5 (TLR5) [34–36]. Vaccines of this design have beentested in the clinic and showed an optimal dose of 1–2 �g in normal

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ealthy adults and about 5 �g in healthy subjects over 65 years ofge. These vaccines were generally well tolerated. They seem to beore immunogenic in the elderly compared to the standard egg-

ased product [36]. The flagellin-fusion approach also seems to helpn the aged mouse model [37].

The other advantage of the E. coli system is speed and capacity.hese bacteria double every thirty minutes or less, grow to high titern chemically defined medium, and typically make 20% of their totalell protein as your product. New HA constructs can be made in twoeeks. In the face of a serious pandemic, or for a low-cost vaccine

or large populations, this type of approach becomes attractive.

1. Universal vaccines based on conserved protiens

A universal flu vaccine has been a long-standing desire in thenfluenza community. The notion of a flu vaccine that could bedministered once with a booster every 5–10 years, maintainingrotection against mild to severe disease, has attracted the atten-ion of the public, biotech investors, public health officials, andaccine manufacturers over the past decade. Why cannot we comep with something better to replace this 60+ year old vaccine? It isertainly not a question of lack of effort. Several conserved aspectsf the virus have been explored as vaccine candidates.

1.1. M2 ectodomain (M2e)

The conserved ion channel, M2, was discovered back in 1980s a curious second open reading frame on the M protein geneegment [38]. It was conserved [39], it had ion channel activity40] a necessary function, and antibody raised against the surface-xposed ectodomain, M2e, could restrict the growth of influenzairus [41]. This collection of properties makes M2e and attractivearget. There is one major problem. Natural infection rarely elicitsnti-M2e immunity [42], therefore extraordinary measures may beecessary to make this little antigen immunogenic.

A great deal of effort has been invested by several groups thatave produced candidate M2e vaccines since this seems to be suchn obvious target.

Walter Fiers’ group in Ghent made a particulate vaccine basedn the hepatitis B core antigen which can be expressed in bacteriand spontaneously self-assembles. Appending the M2e sequenceo the core antigen monomer yields a small particle with multipleopies of the M2e sequence on the surface [43]. This vaccine wasested in the clinic and was found to be immunogenic, althoughhe data have not been published outside of a CIDRAP commu-ication (sciencedaily.com/releases/2007/07/070718002008.htm).his project has taken up by sanofi [44] for further development44]. The mode of action of anti-M2e antibody depends on Fc-eceptor bearing macrophages.

DynaVax has made a vaccine comprising 8 tandem copies of M2eenetically appended to influenza nucleoprotein, a T-cell antigen,nd conjugated to a CpG-containing oligonucleotide. This vaccineas been the subject of a clinical trial, and both components, M2end NP, were immunogenic.

CYTOS Biotechnology made a HepBcAG-M2e vaccine [45]hat afforded some protection in murine models. This vac-ine was tested in a clinical trial (www.who.int/entity/accine research/diseases/.../Bachmann.pdf) and was showno be immunogenic.

Merck made a candidate vaccine based on the M2e peptide

hemically conjugated to their proprietary outer membrane com-lex (OMPC) carrier [46]. OMPC is a TLR2 agonist as well as being aarticle. The M2e vaccine was tested in a clinical trial as an alum-dsorbed particle at two different dose levels, and in combination

012) 4927– 4933

with an ISCOM adjuvant in a two dose regimen. Best anti-M2e titerswere obtained with the highest dose plus ISCOM.

VaxInnate made an M2e vaccine based on four tandem copies ofthe M2e sequence fused genetically to the C-terminus of Salmonellaflagellin. This vaccine also provided solid protection against a lethalchallenge in mice [47]. This vaccine was tested in a clinical trial [48]in a two-dose regimen. Robust anti-M2e titers were elicited with1 �g or less total protein per dose. In a follow-up study, the M2evaccine was co-delivered with traditional TIV [49]. The immuneresponse against M2e was unaffected by the co-delivery of TIV, andthe immune response against the two type A components was aug-mented by about 50%. This suggests that an M2e vaccine may be auseful adjunct for seasonal vaccination.

12. Other conserved antigens

The stalk of HA is relatively conserved, compared to the glob-ular head. Until recently, little attention has been paid to the HAstalk. That has now changed in the wake of discoveries of broadlyneutralizing monoclonal antibodies that bind to the stalk and inter-fere with infection [51–53]. The stalk structure that binds to theseantibodies is involved in fusion with the endosomal membrane andappears to be a pH sensitive intermediate. If one could stabilize thisstructure and use it as a vaccine, a broadly cross-reactive vaccinemight be feasible. One group comprising scientists from the NehruInstitute in India, Merck and Crucell [54] has made a candidate vac-cine, using a battery of molecular modeling techniques, that comesclose to this goal.

Merck also made a vaccine targeting the stalk cleavage site,a poly-basic sequence on the stalk that must be cleaved by ahost protease in order for the stalk to re-orient and penetratethe cell. Synthetic peptide was chemically conjugated to OMPC, asabove. The OMPC–peptide conjugate underwent extensive preclin-ical testing [50] in mice and non-human primates, but this vaccinewas not taken into clinical trials.

Two other, simpler, stalk antigens have been proposed, one ofwhich dates back to 1982 [55]. Palese and colleagues removedthe globular head from virus particles by proteolytic cleavage. Theremaining HA2 stalk revealed cross-reactive HA2 eptiopes. Thisidea was revisited recently [56] using the straight alpha helix,aa76–130, as an immunogen, affording modest protection in amurine challenge. Alternatively, the same lab engineered a “head-less HA” stalk that afforded good protection in a mouse challengemodel [57] when presented as a particle based on an HIV gag core,delivered as electroporated DNA. The stalk with its more conservedstructure remains a seductive target.

13. Back to live viruses

The original live attenuated influenza viruses were attenuatedby multiple serial passages in eggs at low temperature, around30 ◦C, selecting for variants that can grow in the cool nasopharynxbut cannot grow in the lung. We now know enough about the virusand have the tools to manipulate its genetics in a more directedmanner. The non-structural protein NS1 gene segment encodes twoproteins that serve to highjack the host cell’s synthetic machinery[58] by regulating splicing and export of mRNA. Recently, a his-tone like function has been described as further interfering with thehost anti-viral response [76]. Deleting or truncating the NS1 gene[59] has multiple anti-interferon effects that ultimately result in anattenuated virus with potential as a vaccine. Similar NS1 deletions

have been developed by AVIR-Greenhills in Vienna [60]. Furtherengineering at the Mount Sinai-Palese-Garcia-Sastre lab has gen-erated flu viruses that cannot reassert, a feature that should beuseful in the face of a pandemic [61]. This group has also created

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n influenza virus that can carry two HA species [62] using a nine-egment genome. This type of approach, taking advantage of thelasticity of the flu genome, should change the way we think aboutu vaccines.

4. Vaccines intended to elicit T-cell responses

For decades, influenza vaccines have been characterized pri-arily by their ability to elicit hemagglutination-inhibiting (HAI)

ntibody in serum. In preclinical studies we and many others haveeen protection in challenge models in the absence of detectableAI or neutralizing antibody. We assume that a cellular response is

he protective element. Given the repetitive exposure to influenzan the population, infants excluded, the flu vaccines are partlyoosters of preexisting immunity. Adjuvanted trivalent vaccinesave been shown to raise significant Th1 T-cell responses as wells antibody responses [4]. The literature is replete with studieseasuring antibody fold-rise over background and finding that the

ower the background, the higher the fold-rise. The same thingpplies to T-cell responses [63]. Vaccines intended to induce a T-ell response fall into two general categories; vaccines comprisingelected conserved peptides, often as concatamers, and vectoredelivery of antigens. Some labs have combined techniques deliver-

ng multiple selected epitopes via a viral vector or as DNA.

4.1. Peptide based vaccines

An excellent review of T-cell epitope-based vaccine approachesas published just after the 2009 pandemic [64]. We will highlightere specific examples of the various technologies. Ruth Arnon’s

ab has, as mentioned earlier, a concatamer of peptides approachargeting conserved epitopes from NP, M, and HA produced inacteria. Goodman has developed a comprehensive epitope basedn peptides from NP, M1, NS1, PB1, PA, HA and NA expressed in

vaccinia vector [65]. Enhanced CD4 and CD8+ responses werebtained in mice. Alexander has produced a DNA vaccine encoding0 influenza epitopes derived from an array of past and potentialandemic viruses [66] and has demonstrated protection in HLA-DRumanized mice.

4.2. Vectored delivery of antigens

For years, investigators have been using viral vectors (aden-virus, vaccinia variants, alphaviruses, etc.), as a means of attackinghe Big Three pathogens, HIV, TB and malaria [67]. Expression of HAas been achieved in alphavirus vectors [68,69]. Vaccinia vectorsave been used to express the HAs of equine and human influenzairuses [70,71]. Adenovirus vectors expressing influenza antigensave been tested in the clinic [72]. Perhaps the most advanced ofhese approaches is an MVA-based vaccine delivering the NP and

atrix proteins led by Gilbert at Oxford. An initial clinical studyas designed to explore intramuscular and intradermal routes at

ow and high doses [73]. The low dose IM and ID were similar inerms of CD8+ response, while the higher IM dose response was 3-old higher. In a follow-on human challenge study [74], vaccineeshowed a reduced incidence of influenza illness and virus sheddingompared to controls. A similar approach has been used to make aaccine against H5 viruses in the Osterhaus lab [75].

For the field of vectored delivery, one issue that will only beesolved by time and clinical trials is anti-vector immunity. Willt be feasible to administer these vaccines multiple times withoutnterference due to prior exposure to the vector.

[

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15. Where do we go from here?

Clearly, we have a broad range of methods for making influenzavaccines. With an established 60 year old vaccine as the incum-bent, developing a successor, or multiple successors, will take timeand significant effort. Moving away from growing influenza virus ineggs or cell culture will be the first goal. What comes next dependson how well the candidates discussed here fare in the developmentprocess, especially in the clinic and even more so in clinical stud-ies in the elderly where the need is greatest. A critical element forthe success of any of the above programs will be new thinking onthe part of the regulatory agencies regarding the metrics of successin clinical trials of these vaccine candidates. The present guidanceis rooted in the experience with inactivated split virus vaccines,where HAI titers are accepted as correlates of protection for vaccinesof this type. The newer vaccines may require something differentand yet to be defined. Finally, the successful development candi-dates must be easy to manufacture and test for batch release. Thisis not something that is often considered in the early research anddevelopment effort. It is possible to make a wonderful vaccine thatis so complex and costly to produce that it cannot be implemented.

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