REVIEWS

Viruses as vaccine vectors for

infectious diseases and cancer
Simon J. Draper* and Jonathan L. Heeney‡
Abstract | Recent developments in the use of viruses as vaccine vectors have been facilitated
by a better understanding of viral biology. Advances occur as we gain greater insight into the
interrelationship of viruses and the immune system. Viral-vector vaccines remain the best
means to induce cellular immunity and are now showing promise for the induction of strong
humoral responses. The potential benefits for global health that are offered by this field reflect
the scope and utility of viruses as vaccine vectors for human and veterinary applications, with
targets ranging from certain types of cancer to a vast array of infectious diseases.

Reactogenicity
The capacity of a vaccine
formulation to induce an
adverse reaction following
immunization.
*The Jenner Institute,
University of Oxford, Old Road
Campus Research Building,
Oxford, OX3 7DQ, UK.
‡
Laboratory of Viral
Zoonotics, Department of
Veterinary Medicine,
University of Cambridge,
Madingley Road, Cambridge,
CB3 0ES, UK.
Correspondence to S.J.D.
e‑mail: simon.draper@ndm.
ox.ac.uk
doi:10.1038/nrmicro2240
Published online
7 December 2009
Twenty-five years have elapsed since the first published
use of a recombinant virus to deliver antigens from
another infectious agent for the purposes of vaccination.
Vaccinia virus (VACV) recombinant for the hepatitis B
surface antigen (HBsAg) was shown to express HBsAg
and, subsequently, to induce immunity in chimpanzees
that was sufficient to protect against hepatitis B infec-
tion1,2. Despite this promising early start and substantial
further development, the list of licensed viral-vector
vaccines for human use is short, in part owing to the
long, hard road to human licensure (for a review, see
REF. 3). Issues concerning the large-scale production
and stability of such vaccines often require substantial
scientific investment, and stringent safety requirements
must be met for viruses that, in their natural state, have
the potential to be human pathogens. This is especially
true for those viral vectors that remain replication com-
petent or that, by their nature, persist in the host beyond
the inflammatory period. Indeed, in the simplest sense
there remains a range of safety that increases with atten-
uation, which historically had to be balanced with the
belief that reduced replicative capacity would result in
reduced immunogenicity. However, despite these chal-
lenges, arguably the most important factor driving the
continued development of viral-vector vaccines is their
promising immunogenicity, especially with some of
nature’s most difficult antigens belonging to some of the
world’s toughest pathogens and cancers (TABLE 1).
In most cases, a replicating viral infection is very
effective at eliciting robust immune responses in a host
that may last for several years. This is in contrast to many
recombinant antigens that are delivered either as subunit
DNA plasmids or proteins; although these are considered
to be reasonably safe (dependent on the adjuvant), they
have frequently suffered from poor immunogenicity.
Conversely, viral vaccine vectors that replicate in vivo
with a vigour that is similar to their wild-type parental
viruses are often highly immunogenic, but also carry
the risk of recombination, reactogenicity or reversion
to virulence. The search for an optimal medium has
driven renewed effort in the development of attenuated
replication-defective viral vectors, a strategy aimed at
maximizing immunogenicity and safety (FIG. 1).
Attenuating viruses as vaccine vectors
One of the breakthroughs underpinning the viral vac-
cine field over the past decade has been the realization
that replication competence and viral virulence do not
always correlate with immunological robustness. For
example, VACV and its safer, highly attenuated (rep-
lication-defective) cousins — modified vaccinia virus
Ankara (MVA)4–6 and New York attenuated vaccinia
virus (NYVAC)7 — have shown comparable and some-
times even better immunogenicity in animal studies8–11.
Currently, a wide range of virus families are under intensive
development as vaccine vectors for either human or vet-
erinary use, including some that are replication competent
but also many that are specifically attenuated. TABLE 1 out-
lines the main virus families that are under development
as vectors and their associated diseases.
Immune evasion: implications for attenuation.
Historically, the attenuation of viruses has been empiri-
cal, through continuous passage in transformed cell lines
or in atypical hosts, and through delivery by unnatu-
ral routes. Many viruses are complex pathogens, and
large DNA viruses such as poxviruses, adenoviruses
(AdVs) and herpesviruses have devoted much of their

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Table 1 | Viral-vector vaccines in development for human and veterinary use

Recombinant viral Target
vector
Veterinary Human
Adenovirus Avian influenza virus, Plasmodium falciparum, M. tuberculosis, influenza
Mycobacterium tuberculosis and virus, HIV-1 and hepatitis C virus
foot-and-mouth disease virus
Bacteriophage in Shigella None M. tuberculosis
Attenuated canarypox Equine influenza virus, West Nile virus HIV-1 and cancer
virus (WNV), rabies virus, feline leukaemia
virus and canine distemper virus
Fowlpox virus (FPV) Avian influenza virus, FPV and Cancer
Newcastle disease virus (NDV)
NDV Avian influenza virus and NDV None
Turkey herpesvirus Infectious bursal disease virus and None
Marek’s disease virus
Flavivirus YFV-17D WNV WNV, dengue virus and Japanese encephalitis
virus
Lentivirus None Melanoma and HIV-1
Measles virus None P. falciparum and human papilloma virus
Modified vaccinia virus Mycobacterium bovis P. falciparum, M. tuberculosis, influenza A virus, HIV-1
Ankara and colorectal, renal, lung and prostate cancer
New York attenuated None P. falciparum and HIV-1
vaccinia virus
Sendai virus None HIV-1
Vaccinia virus Rabies virus Cancer
YFV-17D, attenuated yellow fever virus strain 17D.

genomes to genes that allow them to evade host immune
responses. Many such viral genes encode proteins that
affect specific immune defences, including those
that act on early innate pathways such as pathways involv-
ing interferons12, pattern recognition receptors (Toll-like
receptors; Tlrs)13,14, chemokines15,16 and cytokines17, as
well as those that act on subsequent adaptive responses
by altering major histocompatibility complex processing
of viral peptides18–20. To date, most processes of attenu-
ation — for instance, through continuous in vitro pas-
sage in cell lines — are non-selective of the types of viral
genes that are lost or become altered. ultimately,
viral variants have been selected for their lack of del-
eterious effects in small animals, such that they are no
longer pathogenic and do the host little or no harm.
However, viral genes that become lost or altered during
the nonspecific attenuation process may not necessarily
be the most optimal in terms of making a virus better for
the immune presentation of recombinant inserts with
a more potent adjuvant effect. Given that many viral
genomes are now well characterized, future efforts to
improve the adjuvant properties of viral vectors and
to engineer them to induce different types of responses
will be based on targeting specific viral genes that dif-
ferentially affect the host’s immune system. For exam-
ple, knocking out viral genes that have evolved to reduce
host antiviral immune responses could reduce immune
evasion properties and improve adjuvant-specific char-
acteristics of certain viral vectors; however, anti-vector
responses may preclude the subsequent re-use of that
vector in an individual.
Empirical versus rational attenuation. Attenuated pox-
viruses were first used during the smallpox eradication
campaign and have since been further developed and
exploited as vaccine vectors. One of these, MVA, was
passaged more than 570 times in chick embryo fibrob-
lasts, lost approximately 15% of its genome and was
ultimately replication defective in mammalian cells4,21,22.
Conversely, NYVAC was derived from the Copenhagen
isolate of VACV by the deletion of 18 different OrFs
through in vitro recombination, which caused replica-
tion deficiency 7. Several recent studies have indicated
that MVA maintains a strong immunostimulatory
capacity, perhaps even greater than that of NYVAC23–26,
whereas a recent primate study of HIV-1 vaccine can-
didates showed that these two vaccinia-derived vec-
tors induced qualitatively different types of immune
responses to the encoded Gag–Pol–Nef (group-specific
antigen–polymerase–HIV-1 negative factor) and enve-
lope (env) antigens — NYVAC induced a CD4+ T cell-
dominant response, whereas MVA induced a stronger
CD8+ T cell response and accompanying CD4+ T cell
responses27. The combined action of many viral pro-
teins, some of which act on Tlr signalling pathways
and other antigen-presenting cell functions, probably
accounts for these differences. until there are better
predictive assays for in vivo outcome, the process will
remain largely empirical and dependant on the best
in vivo models to assess the immunostimulatory prop-
erties of each vector. Nonetheless, in the case of empir-
ically attenuated MVA, the rational deletion of genes
can further enhance immunogenicity, with two reports

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Genetic complexity and replication potential
Alum,
HPV and
MF59,
AS01 and
West Nile
virus
CpG
Adjuvants Virus-like
particle
Ex vivo interferon‑γ ELISPOT
An ELISPOT (enzyme-linked
immunosorbent spot) assay
that is used to measure
antigen-specific effector T cell
responses. Typically,
splenocytes or peripheral
blood mononuclear cells are
incubated with the antigen
(peptide or protein) for
18 hours, and the cytokines
(typically interferon-γ) that are
released during that time by
antigen-specific cells are
captured by a monoclonal
antibody, thereby allowing
enumeration by a modified
enzyme-linked immunosorbent
assay (ELISA) technique.
Cultured interferon‑γ
ELISPOT
A modified ex vivo interferon-γ
(IFN-γ) ELISPOT (enzyme-linked
immunosorbent spot) assay
that involves a prolonged
period of in vitro culture of
lymphocytes before the IFN-γ
ELISPOT assay. Typically, cells
are cultured for 10 days in the
presence of antigen and
supplemented with
interleukin-2. Evidence
indicates that a different
population of T cells, most
likely central memory T cells
that differentiate into effector
T cells during the culture
period, are measured by this
assay, as compared with the
measurement of circulating
effector T cells that are
quantified by the immediate
ex vivo ELISPOT.
Heterologous prime–boost
Repeated immunization using
different vaccines; used to
stimulate a better immune
response when a single
application is not sufficient to
induce a protective response.
64 | jANuArY 2010 | VOluMe 8
Smallpox,
Polio (salk) AdV,
VEE and yellow fever,
and MVA and
sindbis measles and
influenza AAV
FluMist
Inactivated Replication- Replicons Live virus
viruses defective vectors
Figure 1 | A range of co-stimulation — from adjuvants
to live viruses. Attenuated replication-defective viral
Nature Reviews | Microbiology
vectors have been developed as part of a strategy to
maximize immunogenicity and safety. They lie in the
middle of a range of complexity and replication
potential, between vaccine technologies at both
extremes that have been licensed for human use.
Although clinical vaccine development requires a
long-term programme of work, the licensure of such
vectored vaccines (if proved to be safe and effective)
remains a wholly attainable and realistic goal. AAV,
adeno-associated virus; AdV, adenovirus; AS01, adjuvant
system 01 from GlaxoSmithKline; HPV, human papilloma
virus; MF59, oil-in-water emulsion adjuvant from Novartis;
MVA, modified vaccinia virus Ankara; VEE, Venezuelan
equine encephalitis virus; VLP, virus like particle.
demonstrating that the deletion of B15R, which encodes
a soluble interleukin-1β (Il-1β) receptor, can increase
the virus-specific memory CD8+ T cell response28,29.
Similarly, by reducing vector-specific gene expression
of a recombinant VACV, the CD8+ T cell response to
the vector itself can be decreased and that against the
transgene consequently enhanced30. Future efforts
are set to continue along these lines, aiming to delete
more genes, alone and in combination, in an attempt
to rationally enhance the immunogenicity of these
viral vectors.
Enhancing immunogenicity
Prime–boost regimes. In the 1990s, excitement sur-
rounded the advent of DNA plasmid vaccines. However,
it soon became apparent that DNA vaccines by them-
selves were insufficiently immunogenic to generate T cell
responses of a magnitude sufficient to protect against
difficult diseases in humans31,32. with advances in the
quantitative T cell assays that are used in clinical trials,
including the ex vivo interferon-γ ELISPOT (enzyme-linked
immunosorbent spot) and cultured interferon-γ ELISPOT
assays as well as multiparameter flow cytometry33,34, viral
vectors resumed centre stage following the demonstration
of their enhanced potency for cellular immune responses
compared with other vaccine types35,36. However, unlike
DNA vaccines, after one or two administrations in vivo,
host responses to the structural proteins of the viral vec-
tor itself become self-limiting, leading to diminishing
responses against the vaccine insert when the vector is
used for multiple immunizations. The development of
prime–boost protocols has been required to overcome
these anti-vector responses while maximizing the host
response to the vaccine insert.
© 2010 Macmillan Publishers Limited. All rights reserved
Initially, heterologous prime–boost protocols with
common vaccine inserts often used a DNA plasmid to
prime the immune system; however, more recently inter-
est has grown in the use of combinations of different
recombinant viral vectors and in how their sequence of
administration can influence the nature of the induced
immune response. Multiple approaches have now been
tested in both animal models and humans, including
DNA–MVA, DNA–NYVAC, fowlpox virus (FPV)–MVA,
influenza–MVA, AdV–MVA, heterologous AdV–AdV,
and DNA–Sendai virus, targeting a wide range of diseases
from malaria, HIV-1, tuberculosis (TB) and hepatitis C
through to cancers (TABLE 2). A consistent observa-
tion throughout all of these studies is the differential
ability of certain vectors to prime or boost responses.
DNA vaccines, FPV and influenza virus are good prim-
ing vectors, whereas poxviruses (MVA and NYVAC)
are consistently able to boost T cell responses that are
primed by other means. A large body of data now indi-
cates that, in general, recombinant AdV can prime and
boost T cell and B cell responses remarkably well10,31,37–46.
More recently, a combination of three unrelated vector
modalities expressing the same vaccine insert has been
used47,48. The assessment of these vectors has been aided
by a greater in-depth analysis, in particular using multi-
parameter flow cytometry 34, which allows the dissection
of the different T cell populations — both phenotypically
and in terms of cytokine production and effector func-
tion — that are induced by such different vectors and
regimes27,37,49–53. Correlating the protective outcomes of
‘quality versus quantity’ of the T cell responses that are
induced by these different immunization regimes and
vector strategies remains a challenge to the field.
Enhancing humoral immunity. More recent work has
established the use of prime–boost immunization
regimes to induce B cell as well as T cell responses,
in particular AdV–MVA or heterologous AdV–AdV
regimes39,40. The induction of both arms of the adap-
tive immune response is likely to be beneficial for
protection against pathogens such as malaria para-
sites and many viruses, in contrast to mycobacteria,
for which cellular immunity is probably the domi-
nant protective mechanism. AdV vectors seem to be
able to prime and boost B cell responses, in contrast
to MVA vectors, which are far better able to boost
the response than they are to prime it. This may be a
result of prolonged high-level antigen expression fol-
lowing AdV immunization that is more beneficial for
B cell priming, compared with a short burst of antigen
from MVA that is more suited for B cell boosting 54.
Alternatively, studies on plasmacytoid dendritic cells
have shown that a wave of cytokines, specifically type I
interferon (IFN) and then Il-6, is essential for driv-
ing B cell differentiation into effector plasma cells in
response to influenza virus infection55. Conversely, the
priming of transgene-specific antibody responses by
AdV vectors seems to negatively correlate with type I
IFN production 56,57, which is also associated with
reduced transgene expression58. In the case of MVA,
E3L confers resistance to type I IFN and is essential for
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Table 2 | Viral-vector vaccines and prime–boost immunization regimes in clinical development for human use
Recombinant viral-vector Target pathogen Target antigen Vaccine details Developer clinical phase
immunization regime or disease
HAdV-5–HAdV-5 Plasmodium CSP and AMA1 Monocistronic vectors; NMRC and GenVec I/IIa
falciparum malaria HAdV-5 (del E1,E3,E4);
administered as a mixture
AdCh63–MVA P. falciparum malaria ME–TRAP, MSP1 Monocistronic vectors University of Oxford I/IIa
and AMA1 and Okairòs
HAdV-35 P. falciparum malaria CSP None Crucell I
MVA Mycobacterium 85A BCG–MVA (containing University of Oxford and IIb efficacy field
tuberculosis the gene encoding 85A) Emergent BioSolutions trial (South
Africa)
HAdV-35 M. tuberculosis 85A, 85B and 10.4 Fusion antigen; (r)BCG Aeras and Crucell I
prime, HAdV-35 boost,
AERAS-402
MVA Influenza A virus NP and M1 Fusion antigen University of Oxford I
HAdV-5 Influenza virus NP Intranasal administration GenVec I
Flavivirus YFV-17D (live WNV preM-Env Strain NY99 Sanofi Pasteur II
chimeric virus)
Flavivirus YFV-17D (live Dengue virus preM-Env Strains PUO-359, Sanofi Pasteur II
chimeric virus) (subtypes 1–4) PUO-218, PaH881 and
1228
Flavivirus YFV-17D (live Japanese encephalitis preM-Env Strain SA-14-14-2 Sanofi Pasteur III
chimeric virus) virus pre-registration
DNA–NYVAC HIV-1 Gag–Pol–Nef and Clade C EuroVacc I
Env
DNA–MVA HIV-1 Gag, Pol and Env Clade B GeoVax IIa
DNA–HAdV-5 HIV-1 Gag–Pol–Nef and HAdV-5 (del E1,E3,E4) VRC, NIAID (NIH) and II
Env GenVec
ALVAC containing the gene HIV-1 gp160 and gp120 ALVAC–HIV Sanofi Pasteur and III
encoding HIV-1 gp160, and vCP1521 prime and Global Solutions for
protein gp120 AIDSVAX–gp120 subtype Infectious Diseases
B/E boost
HAdV-5 and HAdV-6 HIV-1 Gag–Pol–Nef MRKAd5 and MRKAd6 Merck I
trigene vaccines
VACV and ALVAC or Cancer CEA TRICOM vectors NIH I/II
FPV with or without (pancarcinoma) co-express B7.1, ICAM1
combination therapy and LFA3
MVA Colorectal, renal and 5T4 (solid tumors) TroVax Oxford Biomedica I–III
prostate cancer
MVA Lung cancer MUC1 TG4010 vector Transgene SA IIb
co-expresses IL-2
Lentivirus Melanoma MART1 TCR immunotherapy Caltech and UCLA I
HAdV-6 and AdCh3 Hepatitis C virus NS3, NS4 and NS5 Fusion antigen Okairòs and the I
‘NSmut’mutation in NS5 University of Oxford
85a, mycobacterial mycolyl transferase 85A antigen; AdCh63, chimpanzee adenovirus serotype 63; ALVAC, attenuated canarypox virus; AMA1, apical-membrane
antigen 1; BCG, Mycobacterium bovis bacille Calmette–Guérin; CEA, carcinoembryonic antigen; CSP, circumsporozoite protein; del, deleted; Env, envelope protein;
FPV, fowlpox virus; Gag, group-specific antigen; gp160, glycoprotein 160; HAdV-5, human adenovirus serotype 5; ICAM1, intercellular adhesion molecule 1; IL-2,
interleukin-2; LFA3, lymphocyte function-associated antigen 3; M1, matrix 1 antigen; MART1, melanoma antigen recognized by T cells 1; ME–TRAP, thrombospondin-
related adhesion protein fused to a multi-epitope string; MSP1, merozoite surface protein 1; MUC1, mucin 1; MVA, modified vaccinia virus Ankara; Nef, HIV-1
negative factor; NIAID (NIH), National Institute of Allergy and Infectious Diseases (US National Institutes of Health); NMRC, US Naval Medical Research Center NP,
nucleoprotein antigen; NS, non-structural protein; NYVAC, New York attenuated vaccinia virus; preM, pre-membrane protein; (r)BCG, recombinant BCG; TCR,
T-cell receptor; TRICOM, triad of costimulatory molecules; UCLA, University of California, Los Angeles (USA); VACV, vaccinia virus; VRC, Dale and Betty Bumpers
Vaccine Research Centre; WNV, West Nile virus; YFV-17D, attenuated yellow fever virus strain 17D.

sustained growth in chick embryo fibroblasts, and an
E3L-knockout virus induces enhanced levels of chicken
type I IFN and Il-6 in cell culture59,60. If B cell priming
by MVA occurs in a similar manner to that induced by
the influenza virus, the E3L-mediated immune-evasion
mechanism could explain the weak B cell priming that
is observed with this vector. ultimately, a much better
understanding of how cytokines can affect the induc-
tion of B cell responses by different virus families is
essential to improve the rational design of vectors and
prime–boost strategies that can be tailored to induce
optimal antibody responses.

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Circumventing pre-existing immunity. Viruses that
commonly infect the human population bring with
them the problem of pre-existing immunity. For cer-
tain viral vectors, such as those based on human AdVs
and herpesviruses, this can be a major obstacle. For
example, a candidate HIV-1 vaccine based on the
human AdV serotype 5 (HAdV-5) vector was recently
assessed in a Phase II trial that was conducted in
individuals who were at high risk of HIV-1 infection.
The trial was halted owing to lack of efficacy, but at
the time a nonsignificant trend was observed towards
increased acquisition of HIV-1 infection in those indi-
viduals with higher titres of pre-existing HAdV-5-
neutralizing antibodies at the time of enrollment 52,61.
This issue provoked intense debate about the use of
this commonly occurring human virus as a vaccine
vector, but despite this setback for the HAdV-5 vec-
tor platform other strategies are actively being pursued
to provide viable alternatives that can overcome pre-
existing vector immunity 62. These include modifying
the vector to express heterologous adenovirus hexons63
or using human adenoviruses of rarer serotypes40,44,64,65 or
those from other species such as chimpanzees 66–70.
Importantly, all such new vectors, especially those
exogenous to a species, will need to clear the regulatory,
safety and manufacturing hurdles.
Empirical versus rational design of vectored vaccines.
with our expanding knowledge of immunology and
viral biology, a new era of science in vaccinology has been
established that has provided a future basis for the rational
development of viral-vector vaccines. Optimizing subunit
vaccine antigen design and understanding vector tropism,
including target antigen processing and presentation
in vivo, is crucial to this approach. In the case of VACV, it
has been shown that the route of administration71, as well
as the poxviral promoter used to drive transgene expres-
sion72, can influence the extent to which antigens are
directly or cross-presented to T cells. Similarly, new data
indicate that orthopoxvirus structural antigens, which
are expressed late in infection and normally prevented
from entering the direct presentation pathway owing to
an abortive infection in antigen-presenting cells, are also
specifically hidden in viral factories, thereby escaping the
cross-presentation pathway and hampering the induction
of poxvirus-specific CD8+ T cell responses73. This is not
the case for foreign antigens driven by poxvirus early or
late promoters, because these antigens are not usually tar-
geted to such poxvirus factories. It is important that the
consequences of such observations for vaccine-induced
T cell responses against the vector and the transgene are
better understood, and these data may begin to explain
why poxvirus vectors such as MVA have been shown to
be effective at re-boosting immune responses in humans
12 months after a previous immunization with the same
vector 74.
‘Engineering’ immunity. In contrast to the formidable
complexities of the anti-vector immunity that is associ-
ated with persistent DNA viruses such as adenoviruses,
engineered lentiviruses devoid of much of their structural
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© 2010 Macmillan Publishers Limited. All rights reserved
antigens have been developed as vectors to deliver genes
to specific cells in vivo for intracellular immunization75.
This is made possible in part by the minimal expression
of lentivirus-vector genes and in part because repeated
in vivo administration (as in traditional immunization) is
not the aim. Indeed, lentiviruses are currently being used
to ‘engineer immunity’ for cancer and HIV-1 (REFS 76,77).
Transduction of haematopoietic stem cells with lentivi-
ruses containing T cell receptor genes has now entered
clinical testing for cancer immunotherapy. Substantial
preclinical progress has also been made using these vec-
tors to transduce human CD34+ cells with B cell recep-
tor genes encoding neutralizing monoclonal antibodies
against HIV-1 (REF. 76). Strategies for targeting lentiviral
vectors to specific cell types, such as skin dendritic cell
(DC) subsets, have also been developed; for example, a
mutated version of the Sindbis virus fusogenic protein
that retains the ability to bind DC-SIGN (dendritic cell-
specific ICAM3-grabbing non-integrin) proteins has
been developed78. The development of further specific
targeting technologies could greatly enhance the rational
design of viral-vector vaccines that induce tailored and
effective immune responses for the prevention of a wide
range of infectious diseases and cancers.
Advances in veterinary vaccines
The most successful applications of viral-vector vaccines
have undoubtedly been in the veterinary field. This is
in contrast to the development of vaccines for human
use, which entails more stringent regulatory require-
ments, step-wise clinical trials in human volunteers and
a longer time to licensure and return on investment.
At least 12 viral-vector vaccines are currently licensed
for veterinary use (TABLE 3), and many more are under
development (for a review, see REFS 79,80). Nonetheless,
these technologies still face an uphill struggle against
competition from classical inactivated vaccines, which
are often easier to produce. The challenges of control-
ling avian influenza in poultry are driven by econom-
ics, and require mass vaccination strategies such as
administration in drinking water, by aerosol spray or by
immunization in ovo81 (FIG. 2). Keeping the costs of these
routes of administration low while maintaining vaccine
efficacy poses considerable challenges for recombinant
viral-vector technologies. One successful approach has
been to use the new recombinant Newcastle disease
virus (NDV), which provides a bivalent method for
the control of two different avian pathogens at once
by in ovo immunization82. New vaccine strategies for
another important and highly contagious veterinary
pathogen, foot-and-mouth disease virus (FMDV) 83,
are being explored using replication-defective HAdV-5.
Protective immunity has been elicited in cattle and
swine 7 days after a single immunization with recom-
binant HAdV-5 expressing the FMDV capsid and pro-
tease antigens84,85. unlike for inactivated, whole-virus
vaccines, the ability to distinguish infected from vacci-
nated animals (DIVA; see BOX 1) using this vector vac-
cine platform, coupled with greatly increased biosafety,
would make this effective technology highly desirable
for FMDV-free countries. Similarly, with respect to
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Table 3 | Viral vector veterinary vaccines licensed for commercial use

Recombinant viral Target pathogen Target Target antigen brand name Distributor
vector species
ALVAC (plus tetanus toxoid Equine influenza virus Horses HA (Kentucky and ProteqFlu-Te (Europe) Merial
and Carbopol adjuvant) Newmarket strains) Recombitek (USA)
ALVAC West Nile Virus (WNV) Horses PreM–Env Recombitek Equine WNV Merial
ALVAC Rabies virus Cats Glycoprotein G Purevax Feline Rabies Merial
ALVAC Feline leukaemia virus (FeLV) Cats Env, Gag–Pol Purevax FeLV Merial
ALVAC Canine distemper virus Dogs HA and F RECOMBITEK rDistemper Merial
ALVAC Canine distemper virus Ferrets HA and F Purevax Ferret Distemper Merial
Fowlpox virus (FPV) Avian influenza virus and FPV Poultry H5 HA Trovac AI H5 Merial
FPV Newcastle disease virus (NDV) and FPV Poultry HN and F Vectormune FP-N Biomune
Vaccinia virus Rabies virus Wildlife Glycoprotein G Raboral Merial
NDV (LaSota strain) Avian influenza virus and NDV Poultry H5 HA NewH5 Avimex
Flavivirus YFV-17D WNV Horses preM-Env of WNV in PreveNile Intervet
(live chimeric virus) YFV-17D backbone
HVT (live chimeric virus) IBDV and Marek’s disease virus Poultry VP2 of IBDV in HVT Vaxxitek HVT + IBD Merial
backbone
ALVAC, attenuated canarypox virus; Env, envelope protein; Gag, group-specific antigen; F, fusion antigen; H5 HA, HA from influenza virus H5; HA, haemagglutinin;
HN, haemagglutinin–neuraminidase protein; HVT, Turkey herpesvirus; IBDV, infectious bursal disease virus; Pol, polymerase; preM, pre-membrane protein; VP2,
viral protein 2; YFV-17D, attenuated yellow fever virus strain 17D.

Pre‑erythrocytic‑stage
malaria
Plasmodium sporozoites
invade hepatocytes and
develop over a period of
6–7 days. This time is known
as the liver or pre-erythrocytic
stage and precedes
subsequent blood-stage
infection. The liver stage is
clinically silent and infected
persons do not show any signs
or symptoms of infection.
Blood‑stage malaria
During the liver stage of
malaria infection, parasites
develop into merozoites in
infected liver cells. These
rupture out into the blood after
a period of 6–7 days and
undergo a continuous cycle of
asexual growth in erythrocytes,
lasting approximately
48 hours.
Spot‑forming unit
(SFU). The ELISPOT assay is
enumerated by a modified
enzyme-linked immunosorbent
assay (ELISA) technique. During
the development stage of the
assay, so-called ‘spots’ are
formed on the membrane of
the plates that are used. After
counting the spots, results are
routinely expressed as SFU per
million cells, given that it is
possible to calculate this from
the number of cells known to
have been used in the assay
important respiratory pathogens of livestock such as
bovine tuberculosis, recombinant HAdV-5 or MVA
vaccine vectors are being explored. These vectors,
expressing the mycobacterial mycolyl transferase 85A
antigen, have both been described as effective boosting
agents for the Mycobacterium bovis bacille Calmette–
Guérin (BCG) vaccine in cattle86–88. Challenge experi-
ments are now required to assess protection with these
BCG prime–vector boost regimes in herds of immu-
nized cattle following direct exposure and also in a
natural transmission setting. If more effective than
the BCG vaccine alone, the use of vectored vaccines
to boost BCG-primed responses could hold promise
for the control of bovine TB, which is estimated to
cost the world approximately uS$3 billion per annum.
Importantly, the cattle TB setting continues to provide
useful immunological insight for the design and testing
of candidate human TB vaccines89.
Vaccines for human infectious disease
Viral-vector vaccines were originally developed to
induce protective T cell responses against intracellular
pathogens. However, to date neither a viral-vector vac-
cine nor a vaccine that acts directly by T cell-mediated
immunity (with the possible exception of BCG) has
been licensed for human use. This situation is com-
pounded by the fact that these new approaches have
been chosen for some of the most complex human
pathogens for which conventional vaccine approaches
have not been effective. These include Plasmodium
falciparum malaria, Mycobacterium tuberculosis and
HIV-1. Nonetheless, substantial progress has been
made with recombinant viral-vaccine technologies. A
list of vectors that are currently in development for
human use is presented in TABLE 2. Impressive levels of
T cell immunogenicity are now being reported for the
first time in human clinical trials — a goal that, despite
extensive efforts, has yet to be realized by DNA plas-
mid vaccine technologies alone. Key to this advance
has been the development of AdV-vectored vaccines
— AdV has a remarkable ability to prime immune
responses against a transgene that can be boosted to
high levels with a second vector that is recombinant
for the same antigen, typically MVA or a second
AdV of heterologous serotype. Similarly, both MVA
and AdV can substantially boost immune responses
primed by other means, for example the BCG vaccina-
tion. A comparison of these two widely used vectors is
provided in TABLE 4.
Malaria vaccines. recent progress has been made
using a mixture of two HAdV-5 vectors expressing the
pre-erythrocytic-stage malaria antigen circumsporozoite
protein (CSP) or blood-stage malaria antigen apical mem-
brane antigen 1 (AMA1)90. Following a single immu-
nization in human volunteers, an impressive mean
response to each antigen of 500–1,000 spot-forming units
(SFu) per million peripheral blood mononuclear cells
(PBMCs) was measured by ex vivo IFN-γ elISPOT.
Depletion studies defined the response as a mixed CD8+
and CD4+ T cell response, with the CD8+ phenotype three
to five times as frequent as the CD4+ phenotype among
IFN-γ-secreting responder cells, as measured by flow
cytometry (M. Sedegah and T. richie, personal com-
munication). In an effort to circumvent the problem
of pre-existing host responses to HAdV-5, recom-
binant adenoviruses of rare human serotypes or from
non-human primates are also under development as
malaria vaccine candidates. Priming and boosting with
heterologous AdVs, such as HAdV-35 and HAdV-11,
is being explored preclinically 44 and, encouragingly,
a recombinant chimpanzee adenovirus serotype 63

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REVIEWS

a antibody responses against blood-stage infection39, as

Intranasal Aerosolized well as T cell responses that were partially effective
+ Mucosal and lung immunity + Mucosal and lung immunity against the preceding liver-stage infection91.
– Safety issues in humans + Mass vaccination with nebulizer or
inhaler or by spraying animals Heterologous poxvirus and DNA–poxvirus immu-
– Safety issues in humans nization regimes established the value of prime–boost
Oral approaches in humans, achieving much stronger
+ Cheap mass vaccination
+ Mucosal immunity Intradermal T cell responses in clinical trials than single-vector
– Stability in the GI tract + Clinically relevant approaches31,38,92. Nonetheless, efficacy was limited,
+ Systemic immunity
– Needle required (new and so Phase IIa malaria challenge studies of these new
Intramuscular needle-free ‘vaccine patch’ AdV-vector vaccines, scheduled for 2009–2010, will be
+ Clinically relevant transcutaneous technologies important. even if successful, these promising vaccine
+ Systemic immunity are in development)
– Needle required candidates will face further challenges, including the
potential problems of high-level pre-existing immu-
Intraperitoneal nity to the HAdV-5 vector in the human (especially
Intravenous
+ Used for some candidate + Used for some candidate
African) population93. Furthermore, it remains to be
cancer vaccines cancer vaccines seen how immunogenic AdV vectors will be in the tar-
– Safety issues –Safety issues get population of young African children. In children
between 1 and 6 years of age, living in areas of high
b malaria transmission, reduced vaccine immunogenic-
ity has been reported in comparison with naturally
In ovo immune African adults and malaria-naive volunteers
+ Mass vaccination of animals
– Labour intensive in developed countries94.

Figure 2 | Possible routes of immunization for human and veterinary vaccines.

Nature Reviews | Microbiology
Potential advantages (+) and disadvantages (–) are shown for various possible routes
of vaccination. a | Intramuscular immunization forms the mainstay approach to human
vaccination, with minor exceptions such as Mycobacterium bovis bacille Calmette–Guérin
(BCG) vaccine that are routinely given intradermally. Work is now progressing to
develop needle-free transcutaneous devices such as patches coated with vaccine that
can be self-applied in the absence of a trained individual — an obvious advantage for
mass and emergency vaccination. Certain cancer vaccines may also be administered
intravenously, but in general the safety concerns remain high for this route. Mucosal
immunity may be enhanced in the gastrointestinal (GI) and respiratory tracts through
the use of oral or aerosolized vaccines. This may enhance protective immunity to
pathogens, for example against tuberculosis infection in the lungs or HIV infection in
mucosal tissue. Recombinant adenovirus human serotype 41 (HAdV-41), a natural
enteric pathogen, may prove to be a more suitable vector for the oral route, given that
it has been reported to stimulate immune responses under acidic conditions127.
However, there is still much work to be done in terms of demonstrating and
maintaining vaccine safety, efficacy, stability and dosing before these routes of
vaccination can be widely used in humans. Keeping the costs of using any of these less
standard routes of administration in humans low while maintaining vaccine efficacy
and, more importantly, safety poses considerable challenges for recombinant
viral-vector technologies, and much proof-of-concept research is still required.
b | Veterinary vaccines also make use of standard parenteral immunization routes
(intradermal and intramuscular), but the use of alternative routes such as administration
in drinking water, by aerosol spray or by immunization in ovo are driven by economics,
less stringent safety requirements in animals and the frequent need for mass
vaccination strategies.
(AdCh63) 70 expressing the pre-erythrocytic-stage
antigen thrombospondin-related adhesion protein
fused to a multi-epitope string (Me-TrAP) followed
by a boost with MVA elicits mean levels of >1,000 SFu
per million PBMCs against the malaria antigen in
human volunteers (A. Hill, personal communication).
Further preclinical studies have also reported the use
of an AdV–MVA regime in mice to induce high-titre
antibody responses and potent T cell responses. This
specific HAdV-5–MVA regime, using vectors that are
recombinant for the blood-stage malaria antigen mero-
zoite surface protein 1 (MSP1), induced protective
Tuberculosis vaccines. Similar to the situation for
malaria, highly promising vectored-vaccine pro-
grammes are under way for human TB. These remain
somewhat hindered in the absence of an experimental
human challenge model, although efforts are under way
to develop M. bovis BCG as a surrogate challenge for
M. tuberculosis in humans86. Consequently, efficacy field
trials remain essential once candidate vaccines have been
selected — a process that is both long and expensive. A
BCG prime and MVA–85A (an MVA vector contain-
ing the gene encoding the mycobacterial mycolyl trans-
ferase 85A antigen) boost regimen is now entering into
a Phase IIb efficacy study in South Africa and has so far
demonstrated high levels of T cell immunogenicity both
in the uK and Africa, and an excellent safety record in
over 500 people including adults, adolescents, infants,
HIV-positive individuals and those who are latently
infected with M. tuberculosis 51,95–97. The TB vaccine field
is also developing AdV-vector candidate vaccines and
using similar approaches to circumvent human anti-
HAdV-5 responses by using rare human serotypes such
as HAdV-35. An HAdV-35 vaccine candidate expressing
a fusion of three TB antigens, 85A, 85B and 10.4 (REF. 98),
can boost T cell responses that are primed by either BCG
or a recombinant BCG in non-human primates99, estab-
lishing an approach that is being tested in humans100. A
mixed CD8+ and CD4+ T cell response has been reported
in Phase I studies in both the uSA and South Africa137,
but the contribution of CD8+ T cell responses to human
immunity against TB remains unclear. For both leading
viral-vector vaccine candidates, preclinical studies are
now actively pursuing the potential for aerosolized rather
than systemic delivery, possibly using nebulizers, to try to
enhance the mucosal immunity that may be more pro-
tective against M. tuberculosis infection50 (FIG. 2). Further
preclinical data from non-human primates are necessary
if we are to address these outstanding immunological
questions.

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© 2010 Macmillan Publishers Limited. All rights reserved


Box 1 | Differentiation of infected from vaccinated animals
In the case of certain livestock infections, effective conventional vaccines are available
but cannot be used without interfering with disease monitoring based on serological
testing. Both natural infection and vaccination result in a seropositive animal. A classic
example is foot-and-mouth disease virus (FMDV) in cattle: although inactivated
vaccines can be used effectively to control disease, they are not used routinely in
‘FMDV-free’ countries, because this would compromise a country’s disease-free status
and lead to substantial economic repercussions for international trade. FMDV vaccines
have been used to reduce the spread of the disease during an outbreak, although the
vaccinated animals must be subsequently slaughtered to enable re-establishment of
that country’s FMDV-free status126. Other examples include bovine herpesvirus type 1
infection of cattle (which leads to infectious bovine rhinotracheitis), classical swine fever
and Aujeszky’s disease (caused by pseudorabies virus or suid herpesvirus 1) in pigs79.
One alternative to prevent this scenario is the development of vaccines that allow
the differentiation of infected from vaccinated animals (DIVA). This is achieved by the
use of subunit vaccines (for example, viral vectors expressing a single antigen from
the pathogen) or the selective deletion of identified genes from conventional
attenuated vaccines. Vaccination and natural infection can therefore be distinguished
serologically using suitable diagnostic assays that identify antibody responses against
an antigen in the vaccine versus those against an antigen that is deleted or absent
from the vaccine, to which the animal is only exposed by natural infection with the
wild-type pathogen.
Flaviviruses. Yellow fever, a viral disease, is re-emerging
in naive humans owing to a shift in its epidemiology. A
highly effective attenuated vaccine (YFV-17D) has been
used in humans for more than 70 years101. with the
development of live attenuated flavivirus vectors based
on YFV-17D, a programme of rational development of
flavivirus vaccines has evolved102 in which the preM–
Env genes, encoding the flavivirus pre-membrane
and envelope proteins, have been replaced with those
from west Nile virus, japanese encephalitis virus or
dengue virus to generate chimeric vectors. It has been
far more difficult to stably express non-flavivirus anti-
gens in these vectors, but insertion of non-flavivirus
epitopes has been reported103, as has the stable inser-
tion of GFP 104. Following extensive and successful
safety and immunogenicity testing in Phase I–III trials,
the chimeric flavivirus for japanese encephalitis may
well be the first viral-vector vaccine to be licensed for
use in humans105.
Influenza virus. The development of a cross-strain
influenza vaccine would be a major and long-sought
achievement, especially given the continued seasonal
emergence of new influenza variants, the current
H1N1 influenza A virus pandemic and the possibility
of an outbreak of highly pathogenic avian influenza.
The induction of protective T cell responses against
conserved internal influenza antigens, rather than
variant-specific antibody responses against surface
haemagglutinin and neuraminidase antigens, is one
approach that may stand a chance of success. Phase I
trials of a new candidate cross-strain influenza vac-
cine are now under way, using an MVA vector that
expresses the highly conserved internal nucleoprotein
and matrix 1 (NP–M1) influenza A antigens. This vec-
tor can boost T cell responses in individuals who are
presumably primed by natural exposure (S. Gilbert,
personal communication). If these T cell responses
NATure reVIewS | MicRobiology
© 2010 Macmillan Publishers Limited. All rights reserved
REVIEWS
are associated with protection, as some evidence sug-
gests106, then a viral-vector vaccine approach could
prove highly cost-effective in comparison with sea-
sonal influenza immunizations aimed at inducing
antibodies against the variant-specific surface coat
antigens.
HIV. Perhaps a greater challenge for vaccine devel-
opment than the seasonal variability and antigenic
drift of influenza virus is the constantly evolving HIV
epidemic. T cell-inducing vaccines for HIV-1 aim to
reduce viral load and CD4+ T cell loss, prolong survival
and reduce transmission107. renewed efforts are focused
on poxvirus regimes using attenuated canarypox
virus (AlVAC), MVA or NYVAC. each of these poxvirus
vaccine vectors has been developed and evaluated
for their ability to induce HIV-1-specific immune
responses in humans. The most recent Phase III
HIV-1 clinical trial used a combination of AlVAC
and AIDSVAX (monomeric HIV-1 glycoprotein 120)
in a large, community-based population in which
the risk of transmission is based on heterosexual life
style. Although the study found an encouraging trend
towards prevention of HIV-1 infection (31.2% effi-
cacy), there was no benefit for post-infection virus
load or CD4+ T cell counts108. In preclinical efficacy stud-
ies in non-human primates, control of virus load and
preservation of CD4+ T cell counts have been reported
when certain pox-based vectors have been used in het-
erologous prime–boost combinations to boost DNA-
primed responses41,48. They are able to induce antibody
responses against env and elicit T cell responses of
varying levels. In humans, T cell responses are of a
mixed CD4 + and CD8 + phenotype and are broadly
multifunctional in terms of cytokine production37,109,110.
Nonetheless, the consensus remains that these vaccine
regimes need to be improved to increase the magnitude
and breadth of the CD8+ and CD4+ T cell responses
against antigens such as Gag and Pol, and to induce high-
titre neutralizing antibodies or high-avidity antibodies
against the native trimeric env protein.
The development of HAdV-5 recombinants for
HIV-1 vaccination produced the most immunogenic
candidate vaccine to date, which was taken forward
into a human efficacy trial, designated STeP. The
HAdV-5 recombinants remained a leading approach
for HIV-1 vaccine developers until the STeP trial
was terminated on the grounds of futility 52,61,111. even
though much stronger T cell responses have been
elicited in malaria and TB prime–boost vaccine pro-
grammes in humans, the failure of the STeP trial was
suggested by some as grounds for dismissal of the
concept of vaccines aimed at inducing T cell-mediated
immunity. encouragingly, however, after some initial
despondency, discussion has refocused on the further
optimization of vaccine immunogens and technolo-
gies62. Indeed, now that far higher T cell induction is
being reported for non-HIV infections, it is time to
match these in the HIV vaccine field. Vaccine develop-
ers for HIV-1 have already begun to focus on other, less
established, chimeric viral-vector technologies. Sendai
VOluMe 8 | jANuArY 2010 | 69
REVIEWS
Table 4 | Comparison of human adenovirus and modified vaccinia virus Ankara viral vectors
Family
genus
Species
Serotypes
Virion
genome
Tropism
cell receptors
Vector generation
insert capacity
Transgene promoters
Key differences in virion, genome, vector tropism, vector generation and design of transgene inserts are described. CEFs, chick embryo fibroblasts; CEV,
cell-associated enveloped virus; EEV, extracellular enveloped virus; HAdV-5, human adenovirus serotype 5; IEV, intracellular enveloped virus; IMV, intracellular
mature virus; NYVAC, New York attenuated vaccinia virus; VA, virus-associated.
Carcinoembryonic antigen
(CEA). An oncofetal membrane
glycoprotein and member of
the immunoglobulin gene
superfamily. CEA is expressed
in fetal gastrointestinal tissue,
but is also overexpressed in
many human carcinomas of
the colon, rectum, breast,
lung and pancreas. CEA can
be used as a serological
marker of malignancy and is a
tumour-associated antigen that
is a widely used target for
cancer vaccines.
70 | jANuArY 2010 | VOluMe 8
Human adenovirus (HAdV)
Adenoviridae
Mastadenovirus
Six species (A–F)
51 HAdV serotypes
60–90 nm, non-enveloped icosahedral particles with a
protein capsid (240 hexons and 12 pentons)
34–43 kb double-stranded DNA with 15 transcriptional
units: E1–E5, IX, IVa2, L1–L5 and VA RNA
Broad tropism
Coxsackie adenovirus receptor, heparin-sensitive
receptor, CD46, CD80 and CD86 (subgroup B)
Genomic plasmid with the deletion of one or more
essential genes (E1, E3 and E4) and the transgene
cloned into the deletion site; the vector is transfected
into a packaging cell line (HEK293 or PER.C6), and a
pure recombinant virus is obtained
~7.5 kb in E1- and E3-deleted HAdV-5
Cytomegalovirus, β-actin and elogation factor 1α from
humans, cytomegalovirus from Mus musculus and
ubiquitin
virus, cytomegalovirus (CMV), reovirus, herpes sim-
plex virus and NDV are currently under evaluation by
a range of groups, including the International AIDS
Vaccine Initiative (IAVI). The recent use of CMV as
a vector has shown that this vector has substantial
efficacy in the rigorous SIVMAC (simian immunode-
ficiency virus of the macaque) rhesus macaque chal-
lenge model, exceeding the suppression of virus load
that was observed with another study using HAdV-5
(REFS 112,113). Alternative strategies will probably com-
bine the most immunogenic prime–boost regimes that
are currently available, such as AdV–MVA or heterolo-
gous AdV–AdV regimes or even triple combinations,
to facilitate the recruitment of both T cell and B cell
responses 41,114. A recombinant HAdV-26–HAdV-5
regime has recently been described that can induce a
T cell response against SIVMAC Gag in rhesus macaques
that is stronger, more polyfunctional and more pro-
tective than the response induced by a homologous
HAdV-5 regime40. Indeed, a mean of >2,000 SFu per
million PBMCs was achieved in these macaques40, in
comparison with around 300 SFu per million PBMCs
on average in volunteers in the HAdV-5 homologous
prime–boost STeP trial52. Future studies will reveal
whether these approaches can consistently improve
on the immunological and viral control bench-
marks that were set by the combination of DNA and
HAdV-5 vaccine vectors in the SIVMAC rhesus macaque
challenge model.
Cancer vaccines. The cancer vaccine field (for com-
prehensive reviews, see REFS 115,116 ) has not only
suffered the problem of inducing strong, durable,
© 2010 Macmillan Publishers Limited. All rights reserved
Modified vaccinia virus Ankara (MVA) Refs
Poxviridae None
Orthopoxvirus None
Vaccinia virus None
Attenuated MVA and NYVAC None
300 x 250 nm, enveloped, rounded or brick-shaped 64,128,
virion of multiple forms: IMV, IEV, CEV and EEV 129
178 kb double-stranded MVA DNA with 193 ORFs 64,130
and 177 genes (including 25 pseudogenes)
Broad tropism
Complex entry mechanisms 64,129,
131,132
Attenuated by passage in CEFs (MVA) or by genetic 28,133,
deletion (NYVAC); the transgene is cloned into a 134
plasmid shuttle vector and the vector is recombined in
cell culture into the parental virus genome; a marker
gene allows selection of the recombinant virus
At least 25 kb in MVA 134,135
Natural or synthetic early and late poxviral 5,10,67,
promoters, for example P7.5, SSP, mH5 and I1L 72,90,
136
effective immune responses (an issue shared with
many infectious-disease vaccines), but has also tried
to do so in the face of the unique and complex chal-
lenge of breaking tolerance against self-antigens —
which are, by definition, either weakly immunogenic
or functionally non-immunogenic in an immuno-
logically compromised environment. Intriguingly,
there seems to be hope for certain cancer vaccines in
specific settings. The use of therapeutic viral-vector
vaccines targeting the carcinoembryonic antigen (CeA),
in combination with local radiation, chemotherapy or
cytotoxic T lymphocyte protein 4 (CTlA4)-specific
monoclonal antibodies, has led to improved survival
in some patients117. In this setting, an ‘antigen cascade’
has been observed, whereby T cell responses to the
CeA were noted not only post-vaccination but also as
de novo responses to other tumour-associated antigens
(TAAs), which were possibly induced following more
effective immune targeting of cancerous cells. MVA
vectors expressing TAAs are also currently in advanced
clinical trials as an addition to first-line therapies for
renal, prostate and non-small-cell lung cancers118–120.
recent immunological analyses have indicated that
there is a significant correlation between the level of
activated natural killer cells at baseline and the subse-
quent efficacy in patients receiving chemotherapy plus
MVA–MuC1–Il-2 for lung cancer 121. The level of acti-
vated natural killer cells could potentially be used as
a predictive biomarker for patients who might benefit
from viral-vector immunization combination therapy.
As with immune-compromising viral infections, the
use of cancer vaccines in a therapeutic setting will
require the re-establishment of a functional and robust
www.nature.com/reviews/micro
 REVIEWS

immune response that can be sustained in a potentially
tolerogenic setting — a setting induced as part of the
immune evasion strategy of some cancers. In this light,
combination therapies hold the greatest hope in the
cancer vaccine field, although preclinical data from
mouse122 and non-human primate123 studies indicate
that AdV vectors expressing candidate TAAs can break
tolerance. It remains to be seen whether candidate
cancer vaccines based on AdV vectors can achieve
even better immunogenicity in human trials than that
observed with poxvirus-based immunotherapies.
Future directions
Most viruses have evolved mechanisms to evade host
immune responses and to modulate the subsequent
inflammatory environment in different ways. These
evasive attributes are contrary to the immunostimula-
tory properties of ideal vaccine adjuvants. However, in
the new era of genomics, specific viral genes encoding
factors with immune-evasive properties are being tar-
geted to enhance these immunostimulatory properties.
Not only is this new science in vaccinology using well-
established viral vectors, but as new viruses are discov-
ered and characterized their utility as vaccine vectors
is also being explored. For instance, investigations are
under way using multiple strategies to find alternative
vectors to circumvent the problem of pre-existing vec-
tor immunity. These vectors, combined with new vac-
cine prime–boost regimes that will induce stronger,
broader and more durable effector responses, offer
promising solutions to immunological problems. The
emergence of many new vector systems must not dis-
tract from the great opportunities to improve current
vectors. Approaches that knock out viral genes and result
in improved antigen presentation, improved adjuvant
effects and intentional skewing of immune responses are
important new lines of investigation. New technologies
include the easier manipulation of poxvirus genomes by
‘recombineering’ in bacterial artificial chromosomes28,124,
and high-throughput transgene insertion into AdV vec-
tors, including the use of bacterial tet operator elements
in the transgene promoter to shut down the transcrip-
tion of transgenes that may be toxic or deleterious to
viral growth125. The use of such technologies will be
adopted for a range of different vector platforms, there-
fore allowing the screening of novel viral vaccine vectors
that remain safe but show improved immunogenicity for
the recombinant vaccine antigens that they deliver.
The development of viral-vector vaccines for use
in animals and humans faces substantial challenges,
both in the scientific and regulatory arenas. However,
these technologies are finally achieving the efficacy
in animals and strong immune responses in humans
that have been sought after for so long, in particular
in the fields of malaria, TB and influenza. The transla-
tion of these new technologies will facilitate vaccine
development against some of the toughest challenges
in the field, such as cancer and HIV-1. The licensure of
replication-deficient viral-vector vaccines is feasi-
ble, given that they lie between the two extremes of
protein-in-adjuvant vaccines and live viral vaccines
— both of which, after considerable effort, have been
licensed before. with the first chimeric flavivirus
vaccine against japanese encephalitis virus pending
regulatory approval, the road is paved for many other
viral-vector vaccines in advanced clinical develop-
ment. The next 25 years will prove a challenging but
exciting time for this renaissance in vaccine science.

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Acknowledgements
We thank A. Hill, M. Friede and G. Nabel for organizing the
recent meeting ‘Viral Vector Vaccines 2008’ in Hinxton, which
stimulated the writing of this Review. We thank A. Hill for
comments on a draft of the manuscript and G.J. Nabel for
providing the drawing on which figure 1 is based. J.L.H. is
currently sponsored by grants from the Bill and Melinda
Gates Foundation, USA, and the National Institutes of
Health, USA.
Competing interests statement
The authors declare no competing financial interests.
DATABASES
Entrez Gene: http://www.ncbi.nlm.nih.gov/gene
B15R | E3L
UniProtKB: http://www.uniprot.org
AMA1 | CEA | CSP | MSP1 | mycobacterial mycolyl
transferase 85A
FURTHER INFORMATION
Simon J. Draper’s homepage: http://www.jenner.ac.uk
IAVI: http://www.iavi.org
All linkS ARe AcTiVe in THe online PDF
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