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Live attenuated vaccines: Historical successes and current challenges

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Live attenuated vaccines against human viral diseases have been amongst the most successful cost effective interventions in medical history. Smallpox was declared eradicated in 1980; poliomyelitis is nearing global eradication and measles has been controlled in most parts of the world. Vaccines function well for acute diseases such as these but chronic infections such as HIV are more challenging for reasons of both likely safety and probable efficacy. The derivation of the vaccines used has in general not been purely rational except in the sense that it has involved careful clinical trials of candidates and subsequent careful follow up in clinical use; the identification of the candidates is reviewed. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
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Review
Live attenuated vaccines: Historical successes and current challenges
Philip D. Minor
n
National Institute of Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom
article info
Article history:
Received 18 December 2014
Returned to author for revisions
29 January 2015
Accepted 17 March 2015
Available online 8 April 2015
Keywords:
Live viral vaccines
Smallpox
Polio
Yellow fever
Measles
Mumps
Rotavirus
Vectored vaccines
abstract
Live attenuated vaccines against human viral diseases have been amongst the most successful cost
effective interventions in medical history. Smallpox was declared eradicated in 1980; poliomyelitis is
nearing global eradication and measles has been controlled in most parts of the world. Vaccines function
well for acute diseases such as these but chronic infections such as HIV are more challenging for reasons
of both likely safety and probable efcacy. The derivation of the vaccines used has in general not been
purely rational except in the sense that it has involved careful clinical trials of candidates and subsequent
careful follow up in clinical use; the identication of the candidates is reviewed.
&2015 The Author. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Contents
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Smallpox.............................................................................................................. 380
Poliovirus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
Yellow fever. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
Measles...............................................................................................................386
Mumps................................................................................................................ 387
Rotavirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
Vectored vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
Discussion and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
Introduction
This review is restricted to vaccines against human diseases
caused by viruses although live vaccines have been successfully used
against a range of human and veterinary viral and bacterial infec-
tions. For example the last case of Rinderpest occurred in Kenya in
2001 making Rinderpest vaccine arguably the most successful live
attenuated vaccine to date on the basis that smallpox vaccine was not
derived from variola and was therefore not strictly speaking an
attenuated vaccine.
While live attenuated vaccines against human viral diseases have
been very successful there are many recurrent issues. The safety and
efcacy of certain mumps vaccines is questionable and the rst
rotavirus vaccine to be licensed was withdrawn when it became
clear that it was associated with intussusception. Some vaccines must
be used in a specic way if they are to be maximally useful. For
example in tropical regions, where exposure to the virus occurs
throughout the year, the live attenuated polio vaccine must be given
in mass campaigns to reduce the susceptible population and interrupt
transmission of the virus. In contrast in temperate climates transmis-
sion is seasonal. Thus routine immunisation at a set age is sufcient to
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/yviro
Virology
http://dx.doi.org/10.1016/j.virol.2015.03.032
0042-6822/&2015 The Author. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
n
Tel.: þ44 1707641000.
E-mail address: Philip.Minor@nibsc.org
Virology 479-480 (2015) 379392
reduce the pool of susceptible individuals during the low transmis-
sion period to such an extent that the virus dies out. Similarly measles
vaccine used in mass campaigns has controlled measles by reducing
the number of susceptibles and thus the circulation of virus. This
bypassesthefailureofthevaccinetoworkintheveryyounginthe
presence of maternal antibody. An imperfect vaccine can be used in
such a way as to achieve disease control.
The list of vaccines considered here is not comprehensive but is
chosen to demonstrate how they have been developed over the
years, and that the principle challenge historically has been to
identify a vaccine strain of the right properties when it appears.
Safe and effective vaccines against chronic diseases such as HIV or
HCV remain to be identied in part because the possibility of using
a vaccine that causes serious disease is unacceptable. Successful
vaccines against other diseases associated with chronic infection,
such as varicella zoster have been developed. The characterisation
of a live vaccine strain in terms of its probable attenuation is often
fairly light until clinical trials begin, so that vaccines are developed
by careful empirical clinical science rather than prior design.
There has been much discussion and scientic interest in
vectored vaccines, for example using adenovirus or poxviruses as
the carriers of a protective antigen. The approach has had limited
practical success, although there are interesting developments in
the avivirus area that could come under this heading.
The rst vaccine to be discussed in this paper is that against
smallpox. Vaccinia is not considered by some an attenuated
vaccine in the strict sense because it is a distinct virus from variola
the causative agent of smallpox and not derived from it. On the
other hand the process followed and all of the issues of potency,
safety and quality control raised provide a forceful model for all
live attenuated vaccines developed since. This includes the devel-
opment of an anti-vaccine lobby.
Smallpox
Smallpox was declared eradicated in 1980 making vaccinia
arguably the most successful human vaccine to date. It had sig-
nicant side effects in most rst time recipients and serious, some-
times fatal, effects in a proportion of individuals.
It was common knowledge in rural communities in the UK in
the 18th century that individuals who had contracted cowpox
were resistant to smallpox, and the farmer Benjamin Jesty delib-
erately inoculated farm hands as a protective measure. He never
wrote an account of the experience nor did he challenge the
recipients with smallpox as Edward Jenner did (Jenner, 1798).
Variolation, rst brought to the UK from Turkey by Lady Mary
Wortley Montagu, involved the inoculation of material from a
smallpox sore into the arm of the patient, and, if done correctly
into the supercial layers of the skin, resulted in only 1% mortality
rather than the up to 3040% found for those infected naturally at
the time. Jenner's experiment on the boy James Phipps was
therefore not quite as appalling as it sounds to modern ears.
Phipps was inoculated with material from a cow infected with
cowpox, which resulted in a lesion indicating infection, and then
seven weeks later with material from a smallpox pustule. The
smallpox inoculation did not result in recognisable lesions and
Phipps was clearly protected. Many other examples are given by
Jenner (1798).
The later history of smallpox vaccine illustrates the need for
quality control in vaccines (Baxby, 2001; Minor, 2012a; WHO, 1966,
2003). No potency assay was used until the start of the 20th century,
when a test in rabbits was introduced. Titration on chorioallantoic
membranes of chicken eggs was not introduced until growth in eggs
had been demonstrated (Lazarus et al., 1937) so throughout the 19th
century when vaccination was in theory compulsory in the United
Kingdom, and later into the twentieth century, the vaccinator had at
best a very rough idea of the quantity of active material in the
vaccine. While the vaccine could produce satisfactory lesions and
soresitdidnotfollowthatthesewereduetothevirus,manybeing
caused by bacterial contamination; the preparations might also be
completely inactive. These issues were raised by Jenner (1798).There
was also a practice of growing the vaccine in human arms as well as
in cows with transmission from person to person. Transmission of
syphilis and other diseases by vaccination sometimes occurred as a
result. There was a certain amount of popular resistance to vaccina-
tion which was understandable in view of the fact that it might not
protect you, could kill you from other causes and was in any case
compulsory. As a result the debate that followed was highly partisan.
At the start of the 20th century however compulsion was dropped
in the United Kingdom and the debate became more rational;
evidence was presented that the vaccine worked in epidemics and
data were produced on the nature and duration of the protection it
afforded (McVail, 1902; Baxby, 2002). In 1965 WHO developed
guidelines to provide criteria to be met by a satisfactory product
(WHO, 1966) and in 1967declared the intent to eradicate the disease
by use of vaccine. The guidelines clearlyhintatthepoorstatevaccine
production was in. There was limited control of the strains to be used
(although there were clear differences between the clinical proper-
ties and adverse event prole of the different strains) and little
harmonisation of the general means of production, or of efforts to
minimise or at least detect contaminating organisms. In 2001 when
there was revived interest in vaccine production because of the
attacks on the World Trade Center in New York and the consequent
fear of bioterrorism, the guidelines were revised (WHO, 2003)and
smallpox vaccine production became modernised. There was no
direct way to establish whether the new production methods gave
rise to an effective product as the disease had been eradicated for 20
years, leaving governments with a difcult choice between the old
reactogenic vaccine of which there were still small stocks of
satisfactory potency, new material manufactured by the same
process which involved scarication of calf anks, or the new
vaccines which were of high but different quality and unknown
efcacy in the eld. The problem remains. The basis of the effective-
ness of the vaccine in use is unclear in modern virological terms.
Smallpox vaccine was developed by human challenge studies
and careful clinical examination. Given that the rst description of
a virus as a lterable infectious agent was in 1898 (Beijerinck,
1898), 100 years after the start of smallpox vaccination, it is not
surprising that the inuence of virology on the early development
of smallpox vaccines was minimal.
Poliovirus
The developing understanding of poliomyelitis has been described
(Paul, 1971; Minor, 1992) and the essential features of its pathogen-
esis, including the gaps in understanding, have been the subject of
reviews (Minor, 1997; Nathanson, 2008). Poliomyelitis has nearly
been eradicated (Minor, 2012b) and the main tool in the global
programme initiated in 1988 (WHO, 1988)hasbeentheliveattenu-
ated vaccine developed by Albert Sabin and used since the early
1960s. Many other vaccines both killed (Salk, 1953)andlivewere
explored in the 1950s and the inactivated vaccine of Salk in its
modern form is playing an increasing role in the end game, mainly
because the live vaccine is based on an attenuated form of the wild
type virulent virus which can change. The development of a live
attenuated vaccine depended on an adequate and widely accepted
understanding of the pathogenesis of poliomyelitis which was not
present in the early period.
Poliomyelitis was rare before the end of the 19th century when
it began to appear in regular major epidemics. The rst of these
P.D. Minor / Virology 479-480 (2015) 379392380
were in Sweden and by 1912 much of the information required to
understand the disease was in place. Poliomyelitis had been
shown to be caused by a lterable agent or virus which could be
transmitted to non-human primates. This model also allowed
infectivity to be titrated (Landsteiner and Popper, 1909). Wickman
had shown by careful epidemiological studies that most infections
were silent and that as well as asymptomatic infection there was
an additional systemic phase which might or might not precede
the neurological disease of poliomyelitis (Paul, 1971). Kling among
others had shown that infectivity could be found in intestinal
contents as well as the nervous tissue of fatal cases (Paul, 1971).
Thus Swedish scientists had data that strongly suggested that
there were different phases of infection, at least one of which was
harmless and probably involved replication in the gut. This is the
accepted view today.
Unfortunately an alternative view emerged in the USA where
Flexner focussed on the neurological aspects which are the most
important features of the disease (Paul, 1971). Basing the model on
work with aviviruses he proposed that transmission was via the
nose and involved infection of the olfactory lobes and subsequent
transport through the brain to the lower spine where the damage
occurred. The model was supported by the ability to transmit
infection by instilling poliovirus into the nose of monkeys; this
might also be an effective if articial way of infecting a human. It
was not clear how the virus travelled from person to person. The
model would make vaccine development difcult as it involved
unmediated access to the brain and is cited by some today as a
prime example of the misleading nature of animal models of
human diseases. Flexner's neurological model dominated the eld
for 30 years. Despite this, experimental vaccines were developed
in the 1930s, based on the principles that Pasteur had used in
developing rabies vaccine. One was allegedly a live attenuated
vaccine, the other a killed non-replicating vaccine (Paul, 1971). One
of them was acknowledged to cause disease in recipients in trials
and there is reason to suspect that the other might have done so
as well.
Development of a safe effective live vaccine depended on
understanding the pathogenesis and virology of poliomyelitis.
Albert Sabin, among others, contributed to the refutation of the
neurological model when he showed that virus was to be found in
large amounts in the gut of fatal cases and that such cases had no
sign of viral replication or damage in the olfactory lobes of the
kind found in the monkey model (Sabin and Ward, 1941). By the
mid 1950s the current view of pathogenesis was in place (Bodian,
1955; Sabin, 1956), involving the infection of the gut following
oral-faecal transmission, invasion of the local lymph nodes,
viraemic spread to other still unknown peripheral sites and
eventual invasion of the CNS. It had been shown that immunoglo-
bulin protected against poliomyelitis and that serum antibodies
were therefore a good and sufcient indicator of protection
(Minor, 1997). Thus a virus strain which can induce neutralising
antibodies in the blood and which is able to grow in the gut but
not the nervous system is a candidate vaccine virus.
Polio occurs in three serotypes such that infection with one
does not provide solid cross protection against another. Three
vaccine strains were therefore required and a great deal of effort
went into identifying non-neurovirulent viruses. The type 1 labora-
tory strain Mahoney was derived from a pool of isolates from
clinically unaffected individuals and remains the standard labora-
tory strain. Li and Schaeffer passaged the virus in monkey testis
and other cell cultures and produced several lineages, one of
which, LSc, was eventually developed into the Sabin type 1 vaccine
strain. The type 2 strain P712 was isolated from the stools of a
healthy child from New Orleans and became the Sabin type 2 strain
and the type 3 strain Leon was isolated from a fatal case in 1937
and had become a standard type 3 laboratory strain before
development by Sabin into a vaccine. The passage history of these
viruses to give rise to the Sabin vaccine strains has been recorded
(Sabin and Boulger, 1973) but the key studies were directed to
demonstrating their lack of neurovirulence and the stability of the
attenuated phenotype when inoculated by multiple routes into a
range of primate models (see for instance Sabin et al. (1954)). A
colossal number of old world monkeys of various species as well as
chimpanzees were used in these studies. Sabin also reported
injecting the type 3 strain into human subjects, demonstrating
that they did not seroconvert, and then feeding them the same
strain and showing that an immune response was generated
(Sabin, 1956). The result was the identication of one strain of
each serotype among many studied considered suitable for wide-
spread use. Each production batch was tested for lack of neuro-
virulence in monkeys although the test was not formalised and
made meaningful until the late 1970s and early 1980s (WHO,
1983; Cockburn, 1988). The vaccine was put into a massive trial in
millions of children in the USSR by Chumakov and licensed in the
USA in 1960.
The incidence of poliomyelitis had fallen by about 95% as a
result of the use of the inactivated polio vaccine developed by Salk.
It was reduced still further by the introduction of the live Sabin
vaccine, which was easier to give and could be relatively easily
produced in large amounts. In the United Kingdom there were
between one and 10,000 cases of polio per year in the 1950s but
one to two per year by the end of the 1960s. While there were
other issues including contamination of the vaccine with viruses
derived from the monkeys that provided the cells in which it was
grown (Shah and Nathanson, 1976) the main consideration in what
follows is the vaccine itself.
It became apparent very soon that cases of poliomyelitis were
temporally associated with the live vaccine and while it was hotly
denied by Sabin the view was that in rare instances the vaccine could
give rise to disease. The issue was obscured by the alleged difculty
of differentiating strains until molecular methods were applied
although there was very little real scienticdoubt(Nakano et al.,
1966; WHO Report, 1981). There were recognised changes in the
phenotype of virus excreted by vaccinees compared to the vaccine
they were given including antigenic changes in the type 1 strain and
increases in the virulence of the type 3 strain (WHO, 1969).
Eventually molecular methods showed beyond reasonable doubt
that the vaccine could cause poliomyelitis (Minor, 1980; Nottay et al.,
1981). The viruses had been derived by passage of viruses that were,
whatever their origin, known to be virulent in animals so the
observation was not surprising. The frequency of the cases was so
low as to be difcult to quantify at the time but a large study in the
USA concluded that the incidence was about one in 500,000 rst
time vaccinees and much lower in the previously immunised
(Nkowane et al., 1987); it could also occur in contacts of vaccinees
proving that the virus could spread. The frequencies of vaccine
associated poliomyelitis for the type 1 strain were about a tenth of
those of the type 2 and type 3 strain combined while in contrast the
virulence of the type 1 strain in monkeys was signicantly higher
than that of the other two serotypes (Marsden et al., 1980; Boulger
et al., 1979). It was also known that hypogammaglobulinemic
patients lacking humoral immunity were at greater risk of disease
if given the vaccine but that some (estimated at about 1% exposed)
would go on to excrete virus for periods measured in years instead of
a few weeks (MacCallum, 1971). By the 1970s polio had ceased to be
a public health problem in most developed countries although as the
incidence was unchanged in the rest of the world vaccination had to
continue.
The continued occurrence of vaccine associated cases meant
that the monovalent components of polio vaccines were tested for
safety batch by batch with the only available test, which involved
monkeys. This was cumbersome, expensive and increasingly
P.D. Minor / Virology 479-480 (2015) 379392 381
ethically questionable. There was therefore interest in under-
standing what was being measured and establishing the molecular
and virological basis of the attenuation of the Sabin vaccine strains
of poliovirus. Leon, the virulent type 3 precursor of the Sabin
vaccine strain was cloned and sequenced and compared to the
Sabin vaccine strain itself. Depending on the origin of the vaccine
studied, there were 10 or 11 differences. Creation and recovery of
recombinant viruses showed that the monkey test detected two
(Westrop et al., 1989) or three (Tatem et al., 1992) mutations that
would attenuate the wild type strain, one in the 5
0
non-coding
region involved in the initiation of protein synthesis (Svitkin et al.,
1990) the second in the capsid protein VP3 which made the capsid
less stable and conferred a temperature sensitive growth pheno-
type (Minor et al., 1989) and the third in VP1 which was rapidly
lost on culture or growth in vaccine recipients. The monkey test
involves the injection of the virus directly into the spinal cord and
therefore assesses neurovirulence in the pure sense and not
necessarily as it would be manifest in vaccine recipients where
the virus would have to move from gut to central nervous system
by the blood stream. When the rst two mutations were speci-
cally reverted to the precursor sequence the resulting vaccine
strain virus was almost but not quite as virulent as the wild type
hinting at additional sources of slight attenuation (Westrop et al.,
1989).
Similar studies were performed with type 2 strains where the
vaccine was compared to an isolate from a vaccine associated case;
here the results concerned the reversion of the vaccine to a
paralytic phenotype, rather than attenuation of a wild type strain,
and again two mutations were particularly identied, one again in
the 5
0
non-coding region, the other in the capsid protein VP1.
Other mutations might also have an effect (Macadam et al., 1991,
1993). Finally studies were performed on the type 1 strain
comparing the vaccine to the Mahoney strain from which it
differed by 57 mutations. Here the result was more complex
(Omata et al., 1986); some effect was seen with a mutation in
the 5
0
non-coding region and there was an effect of mutations in
the capsid region, but these were individually less striking and less
easily detected than for type 3 or type 2. In all strains the
mutations in the 5
0
non-coding region are in a single well ordered
structure, domain 5, which forms a part of the Internal Ribosomal
Entry Site (IRES). They are shown in Fig. 1. The thermodynamic
stability of this structure correlates with the neurovirulence of the
virus (Macadam et al., 1993, 2006).
These ndings identied the principal molecular features that
the monkey test detected; later when transgenic mice expressing
the human polio receptor were developed it was shown that the
same mutations were effective in mice (Chumakov et al., 1992).
Moreover for type 3 at least the percentage of revertants detected
at position 472 correlates well with the virulence or attenuation of
commercial batches of vaccine in both monkeys and transgenic
mice (Chumakov et al., 1991). In both animal models direct
inoculation into the spine circumvents any other aspect of patho-
genesis and it was left to studies in vaccine recipients to try to
establish the signicance of the mutations in the human subject.
After vaccination virus excretion is generally thought to occur
for an average of 30 days during which the virus adapts to the
host. The sequence of events for the type 3 strains isolated from
two children given vaccine containing all three Sabin vaccine
strains is shown in Fig. 2, focussing on the known attenuating
mutations and the temperature sensitive phenotype attributable
to the mutation in VP3 (Minor et al., 1986; Minor, 2012a, 2012b).
Both children were immunised as part of the routine UK schedule
at about three months of age; they excreted type 3 virus for 73 and
50 days respectively. The mutation in the 5
0
non-coding region was
lost completely and almost immediately, by 48 h in child 1 and by
day 3, the time of the rst stool produced, in child 2. This suggests
that this mutation so severely handicaps the virus that it cannot
replicate in the human gut to any great extent. However after this
the excreted virus was essentially unchanged for 11 days in both
subjects, before a number of simultaneous alterations occurred:
the excreted virus became a recombinant between type 3 and type
2 (in other children the excreted virus can be a recombinant
between type three and type 1 (Cammack et al., 1988), the
temperature sensitive phenotype was wholly or partially lost,
and some indication of changes in antigenic sites could be
detected. Other differences may also occur including a second
recombination event a few weeks later. It seems likely that the
changes are a response to the initiation of an immune response
which the virus escapes through increasing its replicative tness
by suppressing the temperature sensitive growth phenotype and
by recombining with other viruses as well as changing antigeni-
cally. This would imply that the immune response in the gut at this
stage is not very strong as it can be avoided largely indirectly; it
would also imply that the temperature sensitive phenotype is not
seriously selected against in the absence of an immune response
because the virus has replicated unchanged for 11 days. Moreover
the loss of the temperature sensitive phenotype tends to be
associated with indirect mutations rather than direct reversion.
The type 2 strain reverts in a broadly similar manner but the type1
strain reverts less rapidly and less completely, even in the 5
0
non-
coding region (Dunn et al., 1990). This is consistent with the view
that the type 1 strain is not so handicapped in its replication in the
gut (or as above in the CNS of animal) and is therefore not under
heavy selection for improved tness.
The type 1 strain is the most virulent of the three types in
animal models (Marsden et al., 1980; Boulger et al., 1979; WHO,
2012) yet it causes the lowest frequency of vaccine-associated
poliomyelitis and the identication of the attenuating mutations
responsible has been the most difcult. If there is little selective
pressure to increase its tness further the type 1 strain should be
more stable in vaccinees than the other types. If there are more
mutations each with a lesser effect then selection to high virulence
will also be more difcult because more mutations will be needed.
Thus paradoxically a more virulent virus can be both more effe-
ctive and less virulent in the vaccinee particularly if it contains
many weakly attenuating mutations. The development of a live
attenuated vaccine can therefore be an extremely subtle and
complicated process and is difcult to approach on a purely rat-
ional basis. In contrast the type 3 strain infects recipients given
trivalent vaccine less often than the others, the mutations have
a more readily detectable effect in animal models and the 5
0
Fig. 1. Mutations affecting the virulence of the three Sabin vaccine strains of
poliovirus. Each has a mutation in Domain V of the 5
0
non-coding region and each
at least one mutation in the capsid region. The types concerned are shown in
paranthesis.
P.D. Minor / Virology 479-480 (2015) 379392382
non-coding mutation at least is selected against more strongly in
the human gut. This is consistent with strong selection against a
strong attenuating effect giving a genetically unstable vaccine.
Poliomyelitis has almost been eradicated from the world
thanks to the programme initiated by the World Health Organiza-
tion in 1988 (WHO, 1988, 2014a). Wild type 2 virus was last
isolated from a case in October 1999 apart from an incident
involving of OPV batches which were probably sabotaged
(Deshpande et al., 2003). At the time of writing wild type 3 virus
was last isolated in 2012 (WHO, 2014a), and the last case of any
type in India was in January 2011, which given the social condi-
tions is a colossal achievement. The last case of wild type
1 poliomyelitis in Africa to date was in August 2014. An outbreak
in Syria in 2014 seems to have been brought under control as there
have been no cases for six months, another extraordinary achieve-
ment given the lethal conditions prevailing. Currently Pakistan has
most of the world's cases of wild type poliomyelitis although there
are some cases in Afghanistan. There is a real if fragile possibility
that poliomyelitis caused by wild type virus is about to disappear.
The success of the programme so far is due to the use of the live
attenuated vaccines in mass campaigns so that transmission of the
wild type virus is broken. However the vaccines can also revert to
virulence in vaccine associated cases, and in healthy recipients the
viruses change freely by mutation and recombination in response
to events. Therefore in regions where vaccine coverage is poor, and
the immunised and non-immunised mix in conditions of sub
optimal hygiene, it is not surprising that viruses can be selected
that will transmit freely from one person to another and that such
viruses cause poliomyelitis. They are termed circulating vaccine
derived polio viruses (cVDPV) and there are many instances of
their occurrence, although given the amount of vaccine used they
occur at a low frequency (Kew et al., 2002). Virus excreted by
hypogammaglobulinemic individuals becomes highly virulent but
does not seem to be as transmissible as cVDPVs although there is
no obvious reason why they should not become so. These viruses
are termed immunodecient vaccine derived polioviruses
(iVDPVs). The vaccine therefore poses a problem for the nal
eradication of polio and this is the nal issue. cVDPVs may be
eradicated by vaccinating properly; not all vaccinated individuals
give rise to transmissible strains. Chronic excreters of iVDPVs
usually stop eventually but some can clearly continue for decades
(MacCallum, 1971; MacLennan et al., 2004) and remain a problem
to be solved.
Polio vaccines illustrate the balance that must be struck
between the ability to grow and the inability to cause disease as
well as the fact that once an individual is infected with a live
attenuated virus the situation is in a real sense out of control. The
long period over which poliovirus is excreted is a factor in this but
it makes it possible to investigate what happens to the virus
over time.
Yellow fever
Yellow fever virus is the archetypal avivirus and is transmitted
by the mosquito Aedes aegypti,asrst hypothesised by Carlos
Finlay in 1881 and shown by experimental transmission to army
volunteers by Walter Reed in 1900 (Frierson, 2010). The virus
causes yellow fever which can have fatality rates of 2080%
depending on the circumstances; while none of Walter Reed's 14
volunteers died a repeat of the experiment in Cuba by John
Guiteras in 42 individuals resulted in severe disease in 8 and three
deaths (Frierson, 2010). Many other laboratory and eld workers
became infected in the course of their studies (including Max
Theiler who developed the vaccine in use today) and several died.
Max Theiler fortunately survived. Yellow fever is now conned
mostly to low and middle income countries but was initially also
present in Europe and the Southern States of America including
Texas and Florida; Cuba was particularly affected. While there has
been much concern over the neurotropism of vaccine strains the
primary cause of death from yellow fever is viscerotropic disease
resulting in jaundice.
In 1927 the virus was transmitted to rhesus monkeys from a
Ghanaian called Asibi and in a separate study from a Senegalese
called Mayali. The Asibi isolate gave rise to the 17D vaccine lineage
which is the only one in use today, while the Mayali isolate was
used to develop the French Neurotropic Vaccine strain (FNV) that
was successfully used well into the 1960s when the high incidence
of vaccine associated encephalitis it induced made it unacceptable.
The monkey model made it possible to demonstrate that human
revert 472
3/2 recombinant
non ts
antigenic drift
3/2/3 recombinant
3’ end change
non ts
other point
mutations
end
75604530
15
0
Days post vaccination
Child 1: evolution of type 3 vaccine strain
Child 2: evolution of type 3 vaccine strain
0102030
revert 472
3/2 recombinant
ts ±
antigenic
drift
3/2/3 recombinant
3’ end change
non ts
other point
mutations
40 50
end
Days post vaccination
Fig. 2. Changes in the virus isolated during the excretion of type 3 polio vaccine after the rst immunisation in two infants aged about three months. Child 1 excreted type
3 poliovirus for 73 days and child 2 for 50 as shown by the horizontal time bar. Changes in known attenuating mutations and phenotypes and other phenotypic and
genotypic changes are indicated.
Reprinted from Minor (2012b). The polio eradication programme and issues of the endgame. J. Gen. Virol. 93:457474.
P.D. Minor / Virology 479-480 (2015) 379392 383
serum from recovered cases was protective and that the killed
virus preparations of the time were not effective as vaccines.
It later played a crucial role in the development of vaccines
particularly with respect to the viscerotropism or neurotropism
of virus strains (WHO, 2010). In 1930 Theiler reported that mice
could be infected with yellow fever by the intracerebral route
Fig. 3. Genealogy of yellow fever vaccine strains. All strains are derived from the Asibi strain and the 176 strain derived from it by passage. The divergence of the different
seed strains is shown.
Reprinted from WHO (2010). Recommendations to assure the quality safety and efcacy of live attenuated yellow fever vaccines. Technical report series 978, Annex 5;
pp. 241314.
P.D. Minor / Virology 479-480 (2015) 379392384
producing a more usable model than the rhesus monkey. Passage
in mouse brain resulted in a virus of increased neurovirulence but
decreased viscerotropism in monkeys and the FNV strain passaged
more than 100 times in mouse brain was the rst yellow fever
vaccine to be used in clinical trials in 1931 (Theiler and Smith,
1937 ). For some time after this the FNV strain was used in the USA
and UK in conjunction with human immune serum to further
attenuate it. In France and Africa the vaccine was given by
scarication without serum but in conjunction with small pox
vaccine.
In 1932 it was shown that yellow fever virus could be grown in
chick embryo tissue and both the Asibi and FNV lineages were
passaged extensively in unsuccessful attempts to lessen their
neurovirulent phenotype. Cultures in which the neurological
tissue had been removed were then used and after 100 passages
in this culture type in addition to the previous 76 passages in chick
tissue the neurovirulence of the Asibi strain was reduced. The
strain is referred to as 17D. Attempts to repeat the process with
FNV or unpassaged Asibi virus failed and the isolation of the strain
that went on to be the basis of all yellow fever vaccines in current
use therefore seems to have been pure chance (Frierson, 2010); no
stocks of the Asibi passage 176 virus (17D) are known to exist
today. The 17DD strain diverged at passage 195 and is currently
used in Brazil. The 17D 204 strain originated at passage 204 and its
derivatives are used in the rest of the world, in lines obtained from
Colombia; one group of 17D 204 derived viruses is used in China, a
second in a group of countries including the USA, France and
originally Australia and the Netherlands. The Netherlands virus in
turn was sent to the Robert Koch Institute in Germany where a
stock was made that became the WHO seed (213-77) and
reference preparation (168-73); this is sometimes referred to as
the 213 lineage. A third group of 17D 204 viruses was used in
Colombia, England and India. The situation is summarised in Fig. 3
(WHO, 2010).
Passage is clearly central to the history of yellow fever vaccine,
and the 17D lineage originated from virus grown in cell cultures
prepared from chick embryos where the neurological tissue had
been removed. Yellow fever vaccines are currently produced in
embryonated hen's eggs, a method developed in the late 1930s
(WHO, 2010; Frierson, 2010). It is difcult to establish from the
existing record which of the passages giving rise to the strains
shown in Fig. 3 were in cell cultures from chick embryos from
which the neurological tissue had been removed, in cell cultures
from whole chick embryos, or in embryonated hen's eggs. It is
known that the WHO preparations 168-73 and 213-77 were
manufactured in embryonated eggs at the Robert Koch Institute.
All of the vaccines in current production and use are considered
to be of equally high safety and efcacy but the biological reason
for this is not clear. There are differences in the sequence of the
different seeds and lineages shown in Fig. 3 which demonstrate
that the viruses are distinct, whatever the clinical signicance of
the differences (Santos et al., 1995; Galler et al., 1998; Hahn et al.,
1987; Rice et al., 1985; Dupuy et al., 1989). Given the origin of the
attenuated strains, passage might be expected to affect the
phenotype. Rapid changes on passage of the Asibi strain in HeLa
cell culture systems leading to an attenuated phenotype have been
reported repeatedly over the years (Hearn et al., 1965; Barrett
et al., 1990) and the properties of yellow fever virus are clearly as
changeable as any other virus. In 1941 there were reports of
encephalitis in recipients of a vaccine a few passages on from the
parent strain (Fox et al., 1942); the conclusion was that some
change had been introduced by passage and that the number of
cultures between parent strain and vaccine should be controlled
and restricted. The seed lot system was introduced in which a
master seed is used to generate a working seed that is used in turn
to generate the vaccine itself. Thus passage is restricted to
controlled levels and vaccine production should be more repro-
ducible. By 1943 the original seed material had been distributed
and passaged in various laboratories and manufacturing sites
world-wide and there were signicant differences between the
respective vaccines in their phenotype in monkeys (Fox and
Penna, 1943). In recent years it has been shown that the WHO
seeds and reference (213-77 and 168-73 in Fig. 3) differ in the
monkey neurovirulence test from other seeds such as that origin-
ally used in England (RF 1815 in Fig. 3) or currently in France or
Senegal (S1 IP/F1 and S2 771-2) (Minor, 2011). This is attributed to
the loss of a glycosylation site in the WHO materials. Its signi-
cance for human safety or efcacy is unknown and the relation-
ships between the laboratory markers, the genomic sequences and
the clinical properties of the vaccine in general remain to be
established. However one phase 3 trial reported that the immune
responses to vaccines made from the WHO seed were superior to
those from vaccines made from one of the other 17D 204 groups
(Pster et al., 2005).
Yellow fever vaccine is a classical product. It is known to be
effective, and in fact had a major effect on the disease in Western
Africa in the 1950s as a result of well executed mass vaccination
campaigns before efforts moved on and the disease returned in
the late 1980s; vaccination in South America is known to be
effective with a different lineage of vaccine. The vaccine has also
been considered extraordinarily safe. Given the origin of the
attenuated phenotype in extensive in vitro passage, the serendi-
pitous occurrence of the desired phenotype at passage 176, the
rapid changes in the laboratory phenotype on limited passage of
yellow fever virus in novel culture systems, the need for a seed lot
system arising from the observation of adverse events in humans
and the genetically heterogeneous nature of the different vaccines
in use it is reasonable to wonder why a live attenuated, genetically
unstable vaccine should be so satisfactory in use.
The situation has been further complicated by reports of
serious adverse events, specically vaccine associated neurotropic
disease (YEL-AND) and vaccine associated viscerotropic disease
(YEL-AVD). In the 1940s cases of encephalitis linked to vaccination
with 17D vaccines were reduced by the introduction of the seed lot
system. In the 1950s however there were still a number of such
cases in infants (produced by vaccines made according to the seed
lot system), and WHO recommended that infants below 6 months
of age should not be vaccinated. All except one of the known cases
recovered fully, and the strain from the fatal case was identical to
the vaccine strain antigenically and by the molecular markers
available at the time (Jennings et al., 1994) although it was more
virulent in intranasal mouse neurovirulence tests. YEL-AND was
therefore always a known but rare adverse event whose origin is
not clear. Since 2000 however there have been increasing numbers
of reports particularly in the elderly. The incidence varies depend-
ing on the study from 0.19 to 0.8 per 100,000 doses in Europe and
America (WHO, 2010). Its cause has not been established, speci-
cally whether it is a property of the virus or the host; it is
associated with both 17D 204 and 17DD lineages at similar rates so
far as can be seen.
YEL-AVD or vaccine associated viscerotropic disease, was rst
reported in 1975 in Brazil (Barrett and Teuwen, 2009), and up to
2009 51 cases had been identied. There is no evidence that the
syndrome is caused by changes in the vaccine virus in recipients
(Engel et al., 2006) and host factors such as immunodeciency are
usually quoted. A cluster of ve cases, four fatal, was reported in
2007 in Peru; the viral genome from one of the fatal cases was
sequenced and shown to be identical to that of the vaccine virus;
the root cause of the incident is unknown (Whittembury et al.,
2009). The incidence of YEL-AVD ranges from 0.004 to 0.21 per
100,000 for episodes other than the Peruvian cluster where the
incidence was 7.911.7 per 100,000. The range in frequencies
P.D. Minor / Virology 479-480 (2015) 379392 385
suggests that there may be a problem of ascertainment (WHO,
2010).
The incidence of the adverse events is low but the events
themselves can be very serious or fatal. The reports of YEL-AVD
raised the serious proposal that a major campaign of immunising
against yellow fever in Africa should be abandoned; the risks of
yellow fever were rightly considered greater and the programme
went ahead. It is difcult to know how to proceed when a vaccine
that has been used successfully and safely for decades suddenly
raises concerns. One possibility was that the adverse events were
associated with vaccines of particularly high titre, and this
revealed another issue. The titre of yellow fever virus in vaccines
was dened in WHO requirements in terms of mouse lethality, the
specication being that it should be no less that 3 log
10
mouse
LD50. In practice manufacturers calibrated their mouse test
against a more convenient validated and accurate cell culture
assay. This introduces two sources of possible disagreement
between manufacturers, namely the inaccuracies and variability
of the mouse LD50 test and the similar variability of the cell
culture assay. A study showed that both were very signicant
(Ferguson and Heath, 2004) so that doses of products assayed in
different laboratories were not comparable. Even such a basic
parameter as the amount of vaccine given was thus not known.
The matter has now been corrected by the preparation of an
International Reference preparation calibrating the internal con-
trols used in assays, and the denition of the dose in a common
International Unit (Ferguson and Heath, 2004; WHO, 2010). The
effect of dosage on the incidence of adverse events is not fully
understood but it is clear that if it is a factor it is not the only one.
A better understanding of yellow fever vaccine in its interaction
with the vaccinee would help in the assessment of risks but this is
not the only live vaccine where knowledge is imperfect.
Measles
In developed countries measles is considered a trivial disease of
childhood but in developing countries in the absence of vaccina-
tion the death rate can be as high as 30%; the situation in 19th
century London was similar. In the absence of global vaccination
programmes it is estimated that 6 million children would die of
measles per year, mostly of pneumonia, other respiratory compli-
cations or diarrohea. The vaccines developed in the 1960s to 1980s
are therefore life saving additions to immunisation programmes.
Measles is a complicated disease and a normal infection with
recovery within weeks causes prolonged immune disruption over
a period of a year or more, which for example affects the response
to tuberculosis and some immune mediated syndromes (Moss
et al., 2004). Killed measles vaccines were developed in the 1960s
using a process involving formalin treatment similar to that used
for the manufacture of inactivated polio vaccines; some at least
involved aluminium hydroxide adjuvants which may have been a
factor in what followed. The protection they gave declined in the
medium to long term and when immunised individuals were then
exposed to wild type measles they developed a serious disease
with an atypical rash and a high rate of lung involvement which
could require hospitalisation (Fulginiti et al., 1967). No deaths were
recorded. The susceptibility to atypical measles persisted for many
years; one case was reported 15 years after immunisation
(Fulginiti and Heller, 1980). Initially the aberrant response was
attributed to the nding that the formalin treatment had
destroyed the immunogenic properties of the fusion protein
(Norrby et al., 1975). The consequent absence of antibodies meant
that while cell free virus would be neutralised the virus could
avoid neutralising antibodies by spreading from cell to cell by cell
fusion. Later studies in non-human primate models concluded that
the syndrome resulted from priming for an inappropriate non-
protective type 2 CD4 T-cell response which meant that non-
protective but biologically active anti F protein antibodies were
induced more rapidly than in naïve animals; there was no lack of
antibodies against the F protein (Polack et al., 1999). Atypical
measles raises issues of the suitability of some types of killed
vaccines even today. Similar more serious reactions were also
recorded with vaccines against Respiratory Syncitial virus, another
paramyxovirus, where deaths occurred (Kim et al., 1968). However
despite the subtleties of the immune response the serological
response to measles as measured by neutralising antibody is
accepted as the best marker of protection from infection. Protec-
tive levels have been dened (Chen et al., 1990).
Wild type measles causes fatal acute encephalitis in some
instances. It can also cause subacute sclerosing pan encephalitis
(SSPE) up to 10 years after the original infection, as a result of chronic
virus persistence in the brain of victims. Various genes of SSPE strains
particularly the fusion gene are deleted or modied (Schmid et al.,
1992).TheroleoflivemeaslesinSSPEwasnotdiscovereduntilafter
thedevelopmentoftheliveattenuatedvaccinesandmighthave
caused concern over vaccine use. The vaccines have in fact prevented
many deaths and have never been implicated in SSPE. Measles
vaccines however are clearly a potential mineeld.
The rst isolation of measles virus was described in 1954 (Enders
and Peebles, 1954) and the virus is named the Edmonston strain after
the child concerned. Isolation used primary human kidney and
primary human amnion cells and the virus was subsequently
passaged 12 times in embryonated chicken eggs and 19 times in
primary chick embryo broblast to produce the rst candidate
measles vaccine. The Edmonston B vaccine was derived from this
strain by a further ve passages in chick embryo broblasts at 36
37 1C. It was associated with fever and was initially given with
immunoglobulin to reduce its virulence further; production lots in
cell culture were said to be less reactogenic and were licensed for use
with or without immunoglobulin. Other strains were developed from
the Edmonston strain, and an outline of some of the resulting
vaccines still in global use is given in Fig. 4. In later years there were
attempts to develop a convincing animal model and it was shown
that the isolation of the virus in the usual cell types such as Vero or
human diploid cells gave rise to a virus that would not cause measles
inprimatestothesamedegreeasunpassagedvirus(Kobune et al.,
1996; van Binnendijk et al., 1994). Isolation in peripheral blood
lymphocytes or the marmoset B cell line B95a gave viruses that
were virulent, and also improved the isolation rate, suggesting that
the viruses are more like the wild type, and cells infected in disease
are similar to the lymphocyte lines (Kobune et al., 1990).
The Moraten strain was derived from Edmonston B by 40 further
passages in chick cell culture at 321(Hilleman et al., 1968); the
Schwarz strain by 85 further passages of Edmonston A in chick cells
(Schwarz, 1962) and Edmonston Zagreb by passage of Edmonston B in
the human diploid broblast line WI38 (Ikic et al., 1970, 1972). The
AIK-C strain was derived from the original Edmonston B by growth at
low temperature in chick cells (Hirayama, 1983; Makino, 1983).
Fig. 4. Genealogy of measles vaccines derived from the Edmonston strain.
P.D. Minor / Virology 479-480 (2015) 379392386
Other strains were developed independently from other iso-
lates: CAM 70 from the Japanese Tanabe isolate (Ueda et al., 1970),
Leningrad 4 (the most globally widely distributed measles vaccine
because of its use in WHO programmes) from a Russian isolate,
(Smorodintsev et al., 1960) and Changchun 47 and Shanghai 191
from two independent Chinese strains (Bankamp et al., 2011).
Comparisons of the sequences of the different isolates have been
published which show their relationships (Bankamp et al., 2011).
While the phenotype of the candidate vaccine strains arose
from passage on unusual cell substrates, mainly chicken embryo
broblasts, the question of how a suitable vaccine was recognised
remains; for example the Edmonston B strain was passaged in
chick cells but was initially considered too virulent, causing fever
at an unacceptable rate whereas the Moraten and Schwarz strains
arose from additional passages on the same cell type. Buynak et al.
(1962) compared the phenotypes of the wild type Edmonston
isolate, an independent wild type isolate from Philadelphia and
the Moraten strain in monkeys and in vitro cell culture. The results
were useful in identifying the individual strains but of limited
predictive value for a novel vaccine. The egg passaged viruses, both
Moraten and Edmonston, formed distinctive plaques in chick
embryo broblasts whereas the Philadelphia strain formed pla-
ques only in monkey cells; plaque morphology was distinctive.
None of the viruses caused clinical signs in monkeys when
inoculated by either the subcutaneous of intracranial routes.
Histologically the Philadelphia strain produced lesions when
inoculated intracranially and the original Edmonston strain pro-
duced similar lesions but in only 60% of the animals. The Moraten
strain did not give lesions. The results did not give a clear
indication of the suitability of any of the strains as vaccines but
implied that they were all to some degree attenuated. The obvious
conclusion is that the only way to determine the suitability of the
strains was by clinical trial which is what was done; this was also
the route followed by the Japanese in assessing the AIK-C strain
where several candidates were used and compared for their
clinical side effects (Makino, 1983). Little supportive in vitro
laboratory data is provided in the literature.
While the vaccines in use are life saving and do not have
unacceptable side effects they all cause fever to some extent and
occasional rash about seven days post-immunisation; this resem-
bles mild measles. At some stage they have been accused of more
serious side effects including inducing autism and Crohn's disease
(Duclos and Ward, 1998; Afzal and Minor, 2002; Afzal et al., 2006).
These linkages have not been supported by closer examination but
indicate the mystique of measles and the extent to which it is not
fully understood.
Measlesvaccinesarelivevirusesgivenbyinjectionsothatin
the presence of maternal antibody the vaccine is neutralised and
fails to immunise. In developed countries vaccination is delayed
until about 12 months of age so that maternal antibodies are at
undetectable levels for all recipients. The decay of maternal
antibodies varies between individuals so that some children
may be unprotected for a long time which is a serious risk in
developing countries where mortality rates are high. There have
been attempts to immunise younger babies by using higher titre
vaccines or other strains; immunisation by mucosal routes by
intranasal administration has also been investigated. One obser-
vation that resulted was that the mortality in recipients of
standard dose Schwarz strain was less than in recipients of high
titre vaccines. Death was by causes unrelated to measles and the
effect was particularly seen in girls and in countries where the
death rate was already high. It was suggested that the standard
vaccine had some benecial side effect on the immune system
(Aaby et al., 1993).
In the end the issue of controlling measles with a vaccine that is
not necessarily effective in the main target group of very young
children was solved by giving the vaccine in mass campaigns
regardless of vaccination history (Sabin, 1991). This generates herd
immunity and reduces exposure of susceptible infants. It is an
example similar to that of polio where a vaccine with a aw is
used in a novel way that makes it effective.
Many of the effects of measles vaccine for good or ill are still
unclear. The vaccines have greatly reduced mortality from measles
and in many areas including the Americas indigenous measles has
been eradicated although cases due to imported virus occasionally
occur. It is not necessary to understand how the benecial effects
are generated, but it is an insecure position should some con-
troversy arise. If the cell substrate on which the vaccine is
produced should become suspect the position is difcult for a
vaccine where the cell passage history is crucial, as is the case with
measles. Reverse transcriptase activity was reported in vaccine
produced in embryonic chick cells raising the possibility of
contamination with a retrovirus which might have had serious
consequences for vaccine recipients (Boni et al., 1996). The obvious
solution was to produce vaccine on a different substrate but as the
attenuation of the virus depended on cell passage and only the
Edmonston Zagreb strain was already produced on a different cell
substrate (human diploid cells) in relatively small amounts, this
might well have ended in a non-immunogenic or unacceptably
virulent vaccine or no vaccine at all. In fact the reverse transcrip-
tase activity was associated with defective retrovirus particles
endogenous to the chick embryos at the stage of development at
which they are used for production and is still present in most of
the vaccines used today. It is not thought to pose a hazard
(Robertson and Minor, 1996; Duclos and Ward, 1998).
Mumps
Compared to measles, mumps is not a serious infectious disease
in public health terms, rarely resulting in death. However it is a major
cause of aseptic meningitis in unvaccinated populations and the
parotitis, orchitis and mastitis that are associated with disease are
common, painful and unpleasant, occasionally leading to sterility in
post adolescent males. Mumps vaccine is usually given in combina-
tionwithmeaslesandrubellavaccinesasatripleimmunisation.
Mumpsisthoughttogrowpoorlycomparedtoothervirusesby
those who work on it, there is no reliable marker of protective
immunity as there is for measles or polio and the different assays of
antibody level correlate poorly and give generally low titres (Pipkin
et al., 1999; Yates et al., 1996). Antibodies cross reacting with other
paramyxoviruses including parainuenza are common (Christenson
and Bottiger, 1990). There is no convincing animal model (Parker
et al., 2013) although the disease was transmitted between rhesus
macacques in the 1930s (Johnson and Goodpasture, 1934). Many
strains of mumps have now been sequenced (Jin et al., 2005)and
wild type strains can be classied into different genetic lineages as
with other types of virus including polio and measles. Like these
viruses mumps is so far as is known antigenically monotypic; one
vaccine should protect against all mumps strains. There is evidence
that when the virus is grown on a new cell substrate many mutations
are introduced before the virus grows optimally and while this is true
of other viruses to some extent mumps is a particularly extreme case
(Afzal et al., 2005). It is possible that the right cell substrate to imitate
natural infection has not yet been identied which raises the
questions of what cells the virus grows in in vivo, why it is as
infectious as it is given its laboratory properties and whether
variation in the virus occurs between different in vivo compartments.
The Jeryl Lynn vaccine strain was developed in 1966 from virus
isolated from Maurice Hilleman's daughter, who had developed
unilateral parotitis on March 30th 1963 (Buynak and Hilleman,
196 6). Virus was isolated and passaged in chick embryo amniotic
P.D. Minor / Virology 479-480 (2015) 379392 387
cavity and passaged further on chick embryo broblasts and tested
in a number of institutionalised children; virus at passage 12
produced parotitis in a proportion of recipients while virus at
passage 17 did not. Passage 12 virus also produced higher titres of
neutralising antibody than passage 17 illustrating the trade-off
between attenuation and efcacy. Monkeys were inoculated by the
intrathalamic, intraspinal and intramuscular routes; while they
seroconverted with respect to neutralising antibodies there were
no neuronal lesions or clinical signs with either passage level. No
cell culture system could distinguish the vaccine at different
passage levels from virulent viruses. In short the vaccine was
developed by testing in children without much help from pre-
clinical laboratory studies. Later studies showed a 95% protective
efcacy in children; production was on chick embryo broblasts.
The Urabe strain was developed in Japan by six passages in
chick amniotic cavity, cloning in quail cell cultures and further
passage in eggs (Yamanishi et al., 1970). Six virus clones were
tested in children for neutralising antibody and side effects, and
one clone was selected and designated UrabeAm9. Production was
on chick embryo broblasts. The Rubini strain was isolated from
the urine of a child with mumps, given two passages in human
diploid cells followed by 13 passages in eggs and then further
passages on human diploid cells on which it was eventually
produced commercially (Gluck et al., 1986). The Leningrad Zagreb
strain originated in the Institute for Inuenza in Leningrad, where
it was rst isolated by 15 passages in guinea pig kidney cultures,
ve passages in Japanese quail broblast cell cultures, and addi-
tional passage in guinea pig kidney cells before adaptation to chick
embryo cells in which it was grown for production (Beck et al.,
1989). Other strains have been developed and used (Mrazova et al.,
2003; Boxall et al., 2008).
The Jeryl Lynn vaccine causes mild parotitis in about 1% of
recipients as does the Urabe strain (Balraj and Miller, 1995).
However the Urabe strain also causes aseptic meningitis in a
proportion of recipients about 24 days post-immunisation; the
affected children recover without long term sequelae. The precise
frequency has been difcult to determine because cases were
dened in part by the isolation of Urabe strain virus from CSF
samples obtained by lumbar puncture. This is an invasive proce-
dure and the clinical threshold for performing it is very variable. It
is not clear whether the Urabe strain would be found in CSF of
totally healthy vaccine recipients. The initial ndings suggested a
frequency of aseptic meningitis of 1 in 100,000 in recipients of the
Urabe vaccine, but in the UK this was revised to 17 per 100,000
leading to suspension of the use of the Urabe strain although the
licence was not withdrawn. Figures were higher in other countries
and Japan stopped immunising against mumps altogether. No
increase in the rate of aseptic meningitis was seen in recipients
of the Jeryl Lynn strain although the rate of parotitis was similar to
that seen with Urabe. While the Urabe strain can be isolated from
parotitis cases it appears that Jeryl Lynn cannot.
The Leningrad Zagreb strain was produced in India to supply
the WHO global immunisation programme. It was used in a
campaign in Brazil in 1997 in which 105,098 doses were adminis-
tered to children in ve areas over a period of about 11 weeks.
Fifty ve cases of aseptic meningitis occurred in the weeks
following the campaign, giving a rate of 28.7 cases per 10,000
person weeks, compared to 2.4 in the same period in the previous
year. While it seems reasonable to conclude that the cases were
linked to the vaccine (da Cunha et al., 2002) there was no virus
isolation or characterisation and the obvious conclusion was
disputed by the company, who subsequently performed a study
in Egypt where no link was reported (Sharma et al., 2010). There
were reports of mumps caused by the same strain (Bakker and
Mathias, 2001; Kaic et al., 2008) and of a limited outbreak where
strains from cases were shown to be derived from the Leningrad
Zagreb strain by sequence (Gilliland et al., 2013). The vaccine was
highly immunogenic.
The Rubini strain was used extensively in Switzerland and
Portugal and Singapore (Schlegel and Vernazza, 1998; Afzal and
Minor, 1999; Goh, 1999). Outbreaks of mumps followed but in these
cases the issue was primary vaccine failure where the vaccine was
insufciently immunogenic to afford protection with a single dose.
The vaccine was over attenuated which has also been observed with
other vaccine strains (Mrazova et al., 2003; Boxall et al., 2008).
The balance between attenuation and immunogenicity is
clearly very hard to get right for mumps. However the Jeryl Lynn
strain has not been associated with either aseptic meningitis or
primary vaccine failure although the duration of immunity follow-
ing a single immunisation in early life has been questioned (Cheek
et al., 1995; Miller et al., 1995).
The genomes of isolates from Urabe associated aseptic menin-
gitis were subjected to sequencing and a polymorphism at residue
1081 of the HN RNA was associated with the adverse event (Brown
and Wright, 1998) implying that changes in the virus replicating in
the human host may be of signicance in contrast to yellow fever.
According to existing regulations mumps vaccine seeds must be
tested for neurovirulence by intracranial inoculation of monkeys;
it has been shown that this will not distinguish the vaccine from
the corresponding aseptic meningitis isolate, and in fact while
there are differences between different vaccines and wild type
strains there is no clear correlation with the phenotype in humans
(Afzal et al., 1999). A more sensitive and acceptable form of this
test has been developed in neonatal rats but again while it will
distinguish strains it does not differentiate the vaccine from the
meningitis isolate (Rubin et al., 2005). It is possible that more
sophisticated methods including deep sequencing may help
understanding in particular of the various subpopulations found
(Sauder et al., 2006).
The other vaccines were also subjected to sequencing. The Jeryl
Lynn strain was shown to be a mixture of two very different
strains from the same genetic lineage (Afzal et al., 1993) suggesting
that the original infection involved two distinct viruses, that
contamination had occurred during the isolation and passaging
of the virus or that the viruses had diverged on passage. The
number of differences may be thought to make the last explana-
tion unlikely. The two strains could explain the results of the
original clinical trials if the proportion were related to clinical
phenotype and changed on growth. Secondly the Rubini strain,
reportedly isolated from the urine of a child in Europe, was shown
to be very closely related to the laboratory Enders strain and
distinct from other available European strains (Yates et al., 1996).
The most likely explanation seemed to be that it arose by
laboratory contamination.
Neither of these ndings affected the continued use of the
respective vaccines the decision being based solely on their clinical
performance. They illustrate again the difculty of working with
mumps virus.
Mumps vaccines have a major effect in preventing mumps. It is
disturbing that it is not always clear why, or what properties make
them safe and effective and, in the last analysis, even where they
came from.
Rotavirus
Rotavirus is a member of the family reoviridae, the virion being
composed of 11 segments of double stranded RNA in a complex
capsid. Two of the proteins VP4 (P) and VP7 (G) are targets of
neutralising antibodies; the P protein must be cleaved for the virus
to be infectious. The nomenclature is based on serology and the
genotype of the P protein; thus G1 P7[5] has a G protein that is
P.D. Minor / Virology 479-480 (2015) 379392388
serologically group 1 and a P protein that is serologically group7
and genetically group 5. The rationalised nomenclature of GxPy
was adopted by the working group of the ICTV in 2011; in the
references cited here the order is reversed (PyGx). The segments
can re-assort readily and there are at least 14 G genotypes and 23 P
genotypes. In humans the commonest isolates are G1P8, G2P4,
G3P8 and G4P8 but G9P8 and G9P6 are also isolated. The spectrum
of isolates and disease changes year on year and is very complex
and country dependent (Santos and Hoshino, 2005).
Infections and diarrhoea caused by rotavirus occur world-wide
independent of health or hygiene status because the virus is
excreted in such colossal amounts and is so hardy that disinfection
by conventional methods is more or less impossible. However, the
consequence of infection is very different depending on the region
and country and before vaccination in low or medium income
countries, rotavirus caused 500,000 deaths per year. Irrespective of
the subtype of virus or the location the rst infection is the most
severe; subsequent infections occur but are not life-threatening to
the same extent. Virus excretion at some level is prolonged,
typically 30 days.
In developed countries the mortality rate is very low as a
consequence of available levels of health care. However treatment
such as rehydration is only effective if given in time, which with
the heath care infrastructures in low and medium income coun-
tries can be difcult. Treatment has had a major effect on deaths
from diarrhoea in developing countries but mostly for non-
rotavirus disease (Glass et al., 2005). The introduction of effective
vaccines into existing programmes where high coverage has been
achieved is likely to be one of the most cost effective interventions.
The strategy for developing live attenuated vaccines against
rotavirus has been mixed and efforts continue. Vesikari developed
a vaccine based on an isolate of a calf rotavirus designated
RIT4237, which was a G6P1 type. (Vesikari et al., 1984). This
classical Jennerian approach of taking an agent that resembled
the human virus of interest while not being derived from it gave a
vaccine that worked well in Finland, but failed when tried in
developing countries (De Mol et al., 1986). This was attributed to
the competing effects of other enteric infections; live polio vaccine
was also shown to reduce take of the rotavirus vaccine. Kapikian
used rhesus monkey rotavirus MMU 18006, a G3P3 strain, as the
base but constructed reassortants with MMU 18006 containing the
human G1, G2 and G4 segments to produce the tetravalent vaccine
Rotashield. This was the rst rotavirus vaccine to be licensed
globally in 1998 but was later withdrawn from the market when it
was associated with a low incidence of intussusception in recipi-
ents where the gut invaginates potentially leading to blockage. The
product remains licensed and there is still debate about the
signicance of the syndrome, which may have occurred in those
who were destined to develop it anyway at some time, the vaccine
being the precipitating factor.
Three other vaccines were subsequently licensed: Rotarix,
licensed in Europe in 2006 and in the USA in 2008, Rotateq,
licensed in the USA in 2006 and the Lanzhou strain of lamb
rotavirus, licensed in China and widely used there since 2000.
Rotarix is a single human strain (G1P[8]). Rotateq is a pentavalent
mixture of human/bovine re-assortants (G1P7[5], G2P7[5], G3P7
[5], G4P7[5] and G1P1A[8]). The Lanzhou strain is a monovalent
G10P[12] (Fu et al., 2010). Other vaccines include a monovalent
attenuated neonatal human strain containing one bovine segment
(G9P[11]) manufactured by Bharat in India and other multi- or
monovalent strains in development in many other regions of the
world. The nature of the optimum vaccine is clearly still a matter
of debate with no clear winning strategy. It is not clear whether
multivalent vaccines are needed or whether a monovalent vaccine
could effectively prevent the high mortality associated with the
rst infection by taking the place of the rst attack as some clinical
trials suggest; full protection against infection and disease is strain
specic however. Guidelines for the production of oral rotavirus
vaccines have been developed by WHO (2007) based on the
licensed and effective vaccines in use.
The development of vaccines against rotavirus have thus
followed a familiar pathway of careful clinical evaluation and
unpredicted events.
Vectored vaccines
Vectored vaccines are usually taken to be constructed from a
carrier virus such as an adeno or pox virus which has been
modied to carry a gene from a virus of interest. Thus when the
recipient is given the vector the gene will be expressed and
protective immune responses including antibodies and T cell
responses will be generated. In some cases the relevant gene is
obvious as for the haemagglutinin of measles or the G protein of
rabies virus, although in practice there may be more to protection
than an immune response to a single antigen. Vectored vaccines
have been explored in a number of instances by established
manufacturers (Plotkin et al., 1995; Priddy et al., 2008; Watkins
et al., 2008) and many biotech companies and spinouts. While
the approach has generated licensed gene therapy products and a
rabies vaccine used to immunise wild fox populations (Pastoret
et al., 1995) no licensed human vaccine has resulted. This may be
related to the completeness, duration and protective efcacy of
the immune response generated, commercial concerns about the
scale of production required to meet global requirements and the
availability of easier options that are a priori more likely to
succeed, including the production of live attenuated vaccines or
the non-replicating equivalents. The strategy could lead to
generic solutions to vaccine design if it worked which has not
been clearly demonstrated.
A different category that most would not think of as a vectored
vaccine has been successful however. Yellow fever virus is a
avivirus for which there is a highly safe and effective if rather
poorly understood live attenuated vaccine as reviewed above.
Other human aviviruses include the agents of Japanese encepha-
litis and Dengue fever both of which are major human pathogens
in certain areas of the world. The strategy followed is to replace
the capsid proteins of the 17D derived yellow fever vaccine with
the equivalent region of either JE or one of the four serotypes of
Dengue virus or other aviviruses to give a replicating virus
expressing a novel antigen (Monath et al., 2002). The assumption
is that the attenuated phenotype of yellow fever does not depend
on the capsid region or that replacing it with a foreign capsid
region will itself be attenuating. The JE vaccine Chimerivax based
on this principle has been licensed in Thailand and Australia,
although it is not extensively distributed at the time of writing
(WHO, 2002). The equivalent Dengue construct is in clinical trial
(WHO, 2005, 2014b, 2014c; Capeding et al., 2014). The approach is
virologically less disruptive than the usual vectored vaccine
approaches; most of the machinery remains the same or at least
highly homologous to the natural infection and the vaccine may
therefore be more likely to succeed.
A similar thought process could be applied to live attenuated
inuenza vaccines which use the core of an attenuated virus onto
which the current seasonal surface proteins are grafted by reverse
genetics. Again most would probably not think of this as a vectored
vaccine.
Discussion and conclusions
Live vaccines against viral diseases are one of the most cost
effective health interventions currently available. Their use has
P.D. Minor / Virology 479-480 (2015) 379392 389
eradicated one infectious disease of humans and poliomyelitis is
close to becoming the second. Measles has been controlled in the
Western hemisphere and in much of the developing world by the
use of live viral vaccines and rotavirus vaccines may be the best
way to reduce rotavirus mortality in the world at least in the short
term until better health care infrastructures are developed. Vac-
cines are shown to be successful by clinical experience and
widespread use with monitoring of adverse events and efcacy
as an on-going process.
The understanding of where they came from and why they are
successful is generally poor. The failures of vaccines relate to
adverse events such as causing the disease they are meant to
prevent, or lack of efcacy and there is no clear way to tell where
on the spectrum a given live vaccine lies other than by using it and
acting on the results. For this reason a live vaccine against HIV or
any other virus that causes a lethal disease is problematic, even if
it seems possible that it would be the most likely to succeed. Once
a live vaccine is used it is out of control so far as the patient is
concerned and that can be a challenge.
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In vivo intertypic RNA recombination has previously been observed in excreted type 3 poliovirus isolates from a normal asymptomatic primary vaccinee. This study examines isolates from additional primary vaccinees to determine whether intertypic recombination is a general occurrence in excreted polioviruses. T1 RNase oligonucleotide finger-printing and limited dideoxy primer extension RNA sequencing demonstrated no evidence of intertypic recombination among type 1 or type 2 excreted strains. However, vaccinees excreting type 3 strains for long periods of time eventually produced recombinant strains involving either type 1 or type 2 poliovirus. Moreover, a characteristic time course of appearance of excreted type 3 intertypic recombinant polioviruses was established. Type 3/type 1 and type 3/type 2 recombinant strains appeared at Days 10–11 with a single crossover site in the gene for nonstructural protein 2C. Type 3/type 2/type 3 complex recombinant strains with an additional crossover site in the polymerase gene replaced type 3/type 2 strains at approximately Day 28. A significant portion of the genome of the type 3 Sabin vaccine strain is thus replaced during long-term excretion by vaccinees, and the appearance of some genomic arrangements coincided with a base deletion at the 3′ terminus.
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This document provides guidance to national regulatory authorities and vaccine manufacturers on the production, quality control and evaluation of safety and efficacy of live attenuated rotavirus vaccines (oral) by outlining, in detail, international regulatory expectations for product characterization. It should be read in conjunction with the WHO guidelines on nonclinical evaluation of vaccines (1), and the WHO guidelines on clinical evaluation of vaccines: regulatory expectations (2), in order to understand the whole process of vaccine evaluation. Advice that is specific to the nonclinical or clinical evaluation of live attenuated rotavirus vaccines is provided to supplement these two generic documents. The following text is written in the form of guidelines instead of recommendations. Guidelines allow greater flexibility than recommendations with respect to expected future developments in the field.
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Clinical reaction rates and antibody responses were compared in children given three vaccines: Enders' live, attenuated Edmonston B measles vaccine; a newly described line of Enders' vaccine—live, attenuated Moraten measles virus vaccine; or the live, attenuated Schwarz line of Enders' measles virus vaccine. Groups of 248 to 273 susceptible children in health centers, schools, and outpatient clinics were elected at random to receive the three vaccines. Oral temperature of 101 F (38.3 C) or greater was encountered in 45.9% of those given the Edmonston vaccine, 14.4% of recipients of the Moraten line, and 21.4% of recipients of Schwarz vaccine. The lesser reaction of the Moraten line compared with Schwarz vaccine was statistically significant as was the difference between Moraten and Edmonston vaccine. All children responded serologically to all vaccines. The geometric mean hemagglutination-inhibiting antibody titer following Edmonston vaccine administration was 1:25 compared with 1:16.3 and 1:16.1 for the Moraten and Schwarz vaccines.