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SUPPLEMENT ARTICLE
Increasing Complexity of Vaccine Development
Stanley A. Plotkin
University of Pennsylvania, Doylestown
Vaccines already developed have been enormously successful. However, the development of future vaccines requires solution of a number of immunologic problems, including pathogen variability, short effector memory,
evoking functional responses, and identification of antigens that generate protective responses. In addition, different populations may respond differently to the same vaccine because of genetic, age, or environmental
factors.
Keywords. immune memory; correlates of protection; influenza; pertussis; HIV; rotavirus; dengue; cytomegalovirus; respiratory syncytial virus.
The success of vaccines is counted as one of the crowning achievements of medical science, with enormous
reduction in mortality and morbidity rates [1]. Quoting
the first edition of the Vaccines textbook, “The impact of
vaccination on the health of the world’s peoples is hard
to exaggerate. With the exception of safe water, no other
modality has had such a major effect on mortality reduction and population growth” [2] (Table 1). One of
the great successes of vaccination has been the eradication of smallpox. This was possible because smallpox
was almost always clinically apparent and thus contacts
of a case patient could be vaccinated at exposure. Another success has been the great reduction in the incidence of polio, made possible through the use of 2
vaccines: the inactivated and oral forms. As is well
known, Hilary Koprowski developed the first oral
polio vaccine, although the strains developed by Albert
Sabin ultimately were used worldwide. The 2 vaccines
are complementary, in that the inactivated vaccine
always induces serum antibodies that protect against
paralysis, and the oral vaccine renders the vaccinee
less likely to excrete poliovirus. Nevertheless, despite
the remarkable successes achieved with vaccines, for example against measles, rubella, invasive pneumococcal
Presented in part: Hilary Koprowski Memorial Symposium, The Wistar Institute,
Philadelphia, 27 June 2014.
Correspondence: Stanley A. Plotkin, MD, Vaxconsult, University of Pennsylvania,
4650 Wismer Rd, Doylestown, PA 18902 ([email protected]).
The Journal of Infectious Diseases® 2015;212(S1):S12–16
© The Author 2015. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
[email protected].
DOI: 10.1093/infdis/jiu568
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disease, meningitis, and cervical cancer, there are also
many current failures to develop badly needed vaccines.
This article considers some examples of such failures
and describes the scientific issues responsible for them.
Table 1 lists some of the key problems. The first is
pathogen variability and escape from vaccine-induced
immune responses. This problem limits the efficacy of
influenza vaccines. Although both inactivated and live
vaccines are available, the former is less effective in
infants [3] and the latter less effective in the elderly
[4], but the major problem is that both types are less effective because the circulating viral hemagglutinin often
changes from that used to make the vaccines. Consequently, both vaccines are far from completely effective.
A 2012 meta-analysis [5] showed that influenza vaccine
is less than optimal even in older children and young
adults (Table 2). Its efficacy could be improved in several ways. The first way, namely, the addition of a second type B lineage to make quadrivalent vaccines, has
already been applied. Increasing the dose of hemagglutinin improves antibody responses and therefore efficacy, as does the incorporation of adjuvants into the
vaccine. Adding neuraminidase, which is better conserved than hemagglutinin, might increase efficacy.
However, the optimal improvement would be to add
conserved epitopes to the vaccine. The epitopes might
come from the viral nucleoprotein (T-cell epitopes),
the M2e protein, or, most interestingly, the stalk of
the hemagglutinin. The recent identification of a
conserved epitope far from the variable head of the
hemagglutinin might allow production of a more effective vaccine that would not have to be changed each
year [6].
Table 1.
Key Problems in Vaccinology
Table 3.
Transvaccinology
Better diagnosis
Pathogen variability and escape
Short effector memory
Antigenic changes in current strains
Increased toxin production of current strain
Obtaining the right functional response
Antibodies not bactericidal; efficacy against infection lower than
against disease
Lower efficacy for acellular than for whole-cell pertussis vaccine
(waning antibodies, lower cellular immunity, or fewer virulence
antigens)
Postvaccination immunity wanes faster after acellular pertussis
vaccine
Population specific challenges
Pathogen-specific problems
Uncertain correlates of protection
Unknown antigen(s) required for protection
A second key problem, a rather common one for vaccines, is
short effector memory. This problem is well illustrated by the
recent resurgence of pertussis [7]. Pertussis vaccines made by
inactivating whole bacteria were effective in controlling disease
but were also reactogenic and were therefore replaced by acellular vaccines containing 1–5 pertussis proteins. Although the
acellular vaccines solved the reactogenicity issue, pertussis became resurgent after those vaccines went into standard use in
developed countries. Various hypotheses have been advanced
to explain the resurgence (Table 3). Although strain change
may play a role, the principal problem is the rapid waning of
antibodies, particularly to the pertussis toxin. Current efforts
are directed towards prolongation of immune responses
through the use of adjuvants or more virulence proteins from
Bordetella pertussis [8]. However, an extended duration of effectiveness would be desirable for many vaccines, particularly
those consisting of subunit proteins.
It is the common dogma that antibodies protect by neutralizing entry by viruses into cells and by killing pathogenic bacteria. A means to induce broadly neutralizing antibodies to
human immunodeficiency virus (HIV) before exposure has
bee the subject of intensive research, but with limited success
[9]. Four large phase III trials of AIDS vaccines have been
done, but only one has shown some success [10], the one conducted in Thailand using a canarypox vector carrying the gene
for HIV envelope as prime, followed by the viral envelope protein as boost (Table 4). Protection against acquisition was about
30%, although it was higher early in the trial and then faded,
and it was also higher in low-risk than in high-risk subjects
[11]. The great surprise was that the correlate of protection that
emerged was unrelated to neutralizing antibodies. The major correlate of protection was antibodies against the V1–V2 loops of the
virus functioning as antibody-dependent cellular cytotoxic antibodies [12], particularly of the immunoglobulin G3 class [13].
This finding has also been seen with some other viruses, against
which nonneutralizing antibodies also may have protective functions [14]. The canarypox used for priming in the Thai trial may
also be key to the moderate success of vaccination, because it induces a proinflammatory cytokine milieu.
Vaccine efficacy is also influenced by population-specific
factors. For example, rotavirus vaccines consisting of attenuated
viruses given orally have been highly effective against infantile
gastroenteritis in developed countries, but in poor tropical countries their effectiveness is greatly reduced [15] (Table 5). What is
the explanation for this difference? Possibilities include high levels of maternal transplacental antibodies, neutralization by antibodies in breast milk, interference by simultaneous oral polio
vaccine administration, malnutrition, differences in the intestinal
microbiome, and interference by other intestinal pathogens. The
reasons are incompletely understood and are probably multiple,
but some evidence indicates roles for maternal antibodies and
changes in the intestine due to infections [16].
A vaccine against dengue is a high priority in view of the
spread of mosquito vectors. Neutralizing antibody against the
Table 4.
Table 2.
Adultsa
History of AIDS Vaccines Trials
Efficacy of Influenza Vaccines in Children and Young
Vaccine Type
Dates
Influenza Strain
Efficacy, %
H1N1
74–82
H3N2
B
40–58
51–58
H1N1
86–90
H3N2
B
75–90
44–70
Inactivated
Live
a
Reasons for Pertussis Resurgence
Data from a meta-analysis [5].
Antigen
1998–2003
Envelope gp120
2005–2008
Adenovirus 5 vector
carrying gag
2004–2010
2010–2012
Concept
Result
Antibody
protection
Cellular
immunity
No efficacy
Canarypox vector
carrying envelope,
envelope boost
Prime boost
31% efficacy
DNA for envelope,
adenovirus 5
vector carrying
envelope boost
Prime boost
Negative
efficacy
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Table 5. Rotavirus Vaccine Against Severe Disease in Tropical
Countries
Vaccine
RV1
RV5
Country
Efficacy, %
Brazil
77
Malawi
South Africa
49
77
Nicaragua
77
Kenya
Ghana
83
65
Vietnam
73
Bangladesh
46
envelope protein has been thought to be protective, but a recent
efficacy trial has cast doubt on that idea [17]. The vaccine in the
trial was a quadrivalent live vaccine in which the replicating
virus was the yellow fever 17D attenuated vaccine strain into
which the RNA expressing envelope and premembrane proteins
for dengue types 1, 2, 3, or 4 were inserted [18]. The anticipation
was that each virus would replicate and induce neutralizing antibodies against all 4 serotypes, and indeed early studies showed
that to be the case [19]. Nevertheless, when the vaccine was tested for efficacy in Thailand, although high efficacy was shown
against types 3 and 4, efficacy against type 2 was not seen,
and efficacy against type 1 was only moderate. Several explanations seemed possible, listed in Table 6. Interestingly, recent
data showed that the structure of dengue virus differs according
to the temperature at which virus replication takes place. In cell
culture or in the human at 37°C the structure of the virus is expanded, whereas at lower temperatures in the mosquito the particle is more compact [20]. This could mean that epitopes
exposed on the vaccine virus are not exposed on the mosquito
challenge virus, which is therefore able to enter cells without
being neutralized. However, another explanation for which
there is now evidence is that only viral replication induces homotypic antibodies (N. Jackson, personal communication). In
the quadrivalent vaccine the type 4 chimera replicates well
and induces homotypic antibodies to itself but only heterotypic
antibodies to dengue type 2. The type 2 virus does not replicate
Table 6. Possible Explanations for Low Efficacy of Chimeric
Dengue Vaccine
Higher challenge dose of type 2 or strain variation, so more
antibodies needed
Rapid infection of monocytes by dengue type 2; antibody thus not
effective
T-cell response also needed for protection
Type 2 antibodies heterotypic, not homotypic
Chimera presents of envelope protein in different conformation
Structural differences between virus injected by mosquitoes and
envelope in chimeric virus
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and therefore does not induce homotypic antibodies. The difference between homotypic and heterotypic antibodies may
involve differences in epitopic specificity crucial to protection.
Because human cytomegalovirus (CMV) is the predominant
infectious cause of congenital abnormalities since the elimination of rubella as well as the most common infectious complication of organ and hematogenous stem cell transplantation, a
vaccine against CMV is badly needed [21–23]. The first CMV
vaccine to be extensively tested in humans was a live attenuated
virus developed in my laboratory [24]. This strain, called Towne,
was immunogenic after subcutaneous injection but did not establish latency in vaccinated individuals. Thus, it was safe but
unfortunately in human trials it failed to prevent acquisition
of wild CMV by women exposed to infected children [25].
However, when administered to renal transplant recipients
who lacked prior immunity to CMV and who received a kidney
from a seropositive donor, the vaccine greatly reduced severe
disease [26]. This was the first proof of concept that a vaccine
could influence CMV infection. During that work it was
found that an important antigen for the generation of neutralizing antibodies is a surface glycoprotein called gB [27]. Accordingly, a vaccine was developed consisting of gB together with
MF-59, an oil in water adjuvant [28]. This product was immunogenic after 3 doses, but antibodies fell precipitously thereafter. Importantly, during the first 18 months of a controlled trial
vaccinees had about a 60% reduction of CMV acquisition [29].
Even more striking, the same vaccine reduced viremia in kidney
or liver transplant recipients by >80% [30].
These results were gratifying, but a new discovery changed
our thinking on CMV vaccines: the identification of a pentameric complex of proteins on the surface of the virus, which was
shown to induce the majority of neutralizing antibodies in
human immune globulin. The complex had previously been unnoticed because neutralization tests were done in fibroblast cells,
but CMV can also enter epithelial and endothelial cells by a different mechanism, mediated by the pentameric complex [31].
So it seemed that vaccine developers had been working with
only one of the important CMV proteins [32]. This complex,
composed of the glycoproteins gH and gL together with proteins UL128, 130, and 131, is the subject of intense efforts to incorporate it into next-generation CMV vaccines [33]. Many
candidate CMV vaccines are in development (Table 7).
Respiratory syncytial virus has long been recognized as the
most important respiratory pathogen in infants and second in
importance only to influenza in the elderly. Attempts in the
1960s to develop vaccines against respiratory syncytial virus
have been frustrated first by a disaster with an inactivated
whole virus vaccine that exacerbated disease in vaccinees, second by difficulties in sufficiently attenuating the virus for use
in infants <6 months of age, and third by relatively poor immunogenicity of the fusion protein that induces neutralizing antibodies. Recently, structural biology has come to the rescue of
Table 7.
CMV Vaccines in Development
Table 8.
Unsolved Problems in Vaccinology
Vaccine Type
Problem
Live
Short immune memory
More stimulus of Tfh cells and
stronger induction of innate
immunity by Toll-like receptor
agonists
Multiplicity of virulence antigens
in complex pathogens
Conserved epitopes
Vaccines containing multiple
epitopes
Structural biology to identify
exposed structures
Finding correlates of protection in
protected vaccines
Immaturity and postmaturity of
immune system
Systems biology to identify
unregulated genes
Addition of cytokines or
neutralize cytokines needed
for immune responses
Use of nanoparticles to be taken
up by M cells
Attenuated strain (Towne)
Recombinants with wild virus (Towne-Toledo)
Alphavirus replicon to generate viruslike particles or RNA
Pox virus, adenovirus, lymphocytic choriomeningitis virus vectors
carrying CMV antigens
Replication – defective virus
Nonlive
Recombinant gB glycoprotein with adjuvant
DNA plasmids
Peptides
Viruslike particles
Abbreviation: CMV, cytomegalovirus.
this field by demonstrating a prefusion form of the F protein on
the virus that is highly immunogenic [34]. In addition, adenovirus vectors carrying the F protein are showing promise [35].
Another example of the need to find the right antigens is the
search for a vaccine against group B meningococci. Excellent
vaccines have been available for years against the other major
serogroups but not against group B because of homology between the capsular polysaccharides and neural glycoproteins.
However, the development of genomics, or so-called reverse
vaccinology, permitted the identification of 4 proteins that
induce bactericidal antibodies against most group B strains
[36, 37]. The combination of those proteins with outer membrane vesicle from the organism has been licensed in Europe,
and effectiveness data are awaited [38].
Although the bacille Calmette-Guérin strain of Mycobacterium bovis has been an effective live vaccine against tuberculosis in childhood, its effectiveness against adult tuberculosis
is questionable. Therefore, a new vaccine is sought. Although
candidate vaccines have been developed based on bacille
Calmette-Guérin with added genes from Mycobacterium
tuberculosis, viral vectors carrying those genes, and even
attenuated M. tuberculosis strains, efficacy has been unconvincing [39]. The basic problem is the absence of a known
mechanistic correlate of protection; in other words, the protective immune response is unknown, although it has long
been thought to be a T-cell function [40]. Despite induction
of T-cell responses, a candidate vaccine recently failed to protect in a clinical trial [41].
For a malaria vaccine, the problem is the complexity of the
life cycle and the multiplicity of antigens that would be needed
to give complete protection. Nevertheless, it is clear that the
sporozoite contains antigens that do give protection, notably
the circumsporozoite antigen, and that protection is mediated
by both B-cell and T-cell responses [42, 43]. Injection of
Mucosal immunization with
nonreplicating antigen
Possible Solution
Adjuvants capable of selectively
expanding cell types: dendritic,
B, Th1, Th2, Th17, CD4+,
CD8+, or Treg cells
Use of single or combined Tolllike receptor ligands directed
to specific cell types
Difficulty in generating T-cell
immunity without replicating
vaccines
Addition of adjuvants that
stimulate T cells
Abbreviations: Tfh, follicular T-helper; Th, T-helper; Treg, regulatory T.
whole inactivated sporozoites has shown high efficacy [44].
The task now is to add other sporozoite antigens to improve
on the moderate protection thus far achieved [44, 45].
Stepping back and considering vaccinology as a branch of
immunology, one is forced to realize that there are many unsolved problems, including immune memory, multiplicity of
protective antigens for some pathogens, multiple HLA types
among vaccinees, the need for conserved epitopes, identification of correlates of protection, immaturity of the germinal centers in the newborn, immunosenescence of T cells in the elderly,
and insufficient stimulation of mucosal immunity. Table 8 suggest some pathways to solve these problems during the coming
years.
In conclusion, vaccinology has made great strides in the past
but is now attempting to deal with infections that are complex
and host factors that mitigate efficacy. Fortunately, the advances
in molecular biology, structural biology and systems biology
offer concrete opportunities to solve these difficult problems,
but new vaccines will be the fruit of difficult labor. Hilary Koprowski would have been happy to face these intellectual issues,
and it is a pity that he is no longer with us.
Note
Potential conflict of interest. The author is a consultant to many vaccine manufacturers, including those attempting to develop the vaccines
discussed.
The author has submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
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