Hookworm vaccines: past, present, and future

Article (PDF Available)inThe Lancet Infectious Diseases 6(11):733-41 · December 2006with100 Reads
DOI: 10.1016/S1473-3099(06)70630-2 · Source: PubMed
Hookworms are gastrointestinal nematodes that infect almost 1 billion people in developing countries. The main clinical symptom of human hookworm infections is iron-deficiency anaemia, a direct consequence of the intestinal blood loss resulting from the parasite's feeding behaviour. Although treatment is available and currently used for the periodic removal of adult hookworms from patients, this approach has not effectively controlled hookworm in areas of rural poverty. Furthermore, treated individuals remain susceptible to reinfection following exposure to third-stage infective hookworm larvae in the soil as early as 4-12 months after drug treatment. Therefore, a prophylactic vaccine against hookworm infection would provide an attractive additional tool for the public-health control of this disease. The feasibility of developing a vaccine is based on the previous success of an attenuated larval vaccine against canine hookworm. Several laboratory and field studies have explored the development of a human anti-hookworm vaccine, describing potential protective mechanisms and identifying candidate antigens, one of which is now in clinical trials. The current roadmap that investigators have conceived has been influenced by vaccine development for blood-feeding nematodes of livestock and companion animals; however, recombinant vaccines have yet to be developed for nematodes that parasitise animals or human beings. The roadmap also addresses the obstacles facing development of a vaccine for developing countries, where there is no commercial market.


http://infection.thelancet.com Vol 6 November 2006
Hookworm vaccines: past, present, and future
Alex Loukas, Jeff rey Bethony, Simon Brooker, Peter Hotez
Hookworms are gastrointestinal nematodes that infect almost 1 billion people in developing countries. The main
clinical symptom of human hookworm infections is iron-defi ciency anaemia, a direct consequence of the intestinal
blood loss resulting from the parasite’s feeding behaviour. Although treatment is available and currently used for the
periodic removal of adult hookworms from patients, this approach has not eff ectively controlled hookworm in areas
of rural poverty. Furthermore, treated individuals remain susceptible to reinfection following exposure to third-stage
infective hookworm larvae in the soil as early as 4–12 months after drug treatment. Therefore, a prophylactic vaccine
against hookworm infection would provide an attractive additional tool for the public-health control of this disease.
The feasibility of developing a vaccine is based on the previous success of an attenuated larval vaccine against canine
hookworm. Several laboratory and fi eld studies have explored the development of a human anti-hookworm vaccine,
describing potential protective mechanisms and identifying candidate antigens, one of which is now in clinical trials.
The current roadmap that investigators have conceived has been infl uenced by vaccine development for blood-feeding
nematodes of livestock and companion animals; however, recombinant vaccines have yet to be developed for
nematodes that parasitise animals or human beings. The roadmap also addresses the obstacles facing development of
a vaccine for developing countries, where there is no commercial market.
Hookworm infection and disease through time
“Just how much human helminthiasis is there in the
world? The bare mention of the question will make those
of you with nosogeographical interests—or, better,
helminthogeographical interests—warily scratch a
mental ear and mull over a remark that ends ‘where
angels fear to tread’.”
Human beings and hookworms have a long history
together. There is evidence for hookworm infection in
human beings dating back to pre-Columbian times in
the New World and Pharaonic times in the Old World.
Ancylostoma duodenale was discovered more than
150 years ago, and more than 100 years have elapsed
since Necator americanus was described and the extent of
the problem of hookworm in the southern USA
Around this time, the Rockefeller
hookworm eradication campaign was started in the
southern USA, extended globally in 1913, and nally
terminated in the 1950s.
In his landmark paper, This wormy world, Norman
provided the rst global estimates of the numbers
of people infected with hookworm and showed that
helminth infections were among the most common of
human infections, with hookworm estimated to infect a
third of the world’s population. Substantial regional
economic growth, including improvements in sanitation
and clean water in parts of North America, Asia, and
Latin America have translated into major reductions in
endemic hookworm. For example, economic gains in
the southern states of the USA led to sustained
reductions in the region during the 20th century. Today,
endemic hookworm has been largely eradicated in the
Comparable economic development in several
east Asian countries, including Japan, South Korea, and
Taiwan, during the 1960s and 1970s resulted in sustained
and successful control of hookworm and other nematode
infections, with national prevalence levels currently
below 1%.
In China, rapid economic development has
also led to substantial decreases in the prevalence of
hookworm over the past two decades.
There have also
been well-documented decreases in prevalence in
several Latin American countries, a change largely
attributable to national treatment programmes
coincidental with social and economic development,
which have brought about improved access to clean
water and proper sanitation.
Eff orts to document these changes across the continents
have led to several initiatives to estimate and map the
global prevalence of hookworm and other major soil-
transmitted helminth infections,
with the most recent
estimates provided by de Silva and colleagues.
Here, we
extend this analysis by estimating changes in hookworm
prevalence over the past 60 years. Figure 1 presents a
region-by-region analysis of prevalence estimated from
1947 to the present day. The results suggest that although
Lancet Infect Dis 2006; 6:
Division of Infectious Diseases
and Immunology, Queensland
Institute of Medical Research,
Brisbane, Australia
(A Loukas PhD); Sabin Vaccine
Institute and The George
Washington University,
Washington, DC, USA
(J Bethony PhD,
Prof P Hotez MD); and London
School of Hygiene and Tropical
Medicine, London, UK
(S Brooker DPhil)
Correspondence to:
Dr Alex Loukas, Division of
Infectious Diseases and
Immunology, Queensland
Institute of Medical Research,
Brisbane, QLD 4006, Australia
Tel +61 7 3845 3702;
fax +61 7 3845 3507;
Prof Peter J Hotez, Department
of Microbiology, Immunology
and Tropical Medicine, George
Washington University,
Washington, DC 20037, USA
Tel +1 202 994 3532;
fax +1 202 994 2913;
Latin America
and Caribbean
Prevalence (%)
1994 2003 2006
Figure 1: Prevalence of hookworm infection by region over time
Asia includes China, India, east and south Asia, but not the middle east. Data
from references 1, 12, 16, 19.
http://infection.thelancet.com Vol 6 November 2006
there have been precipitous decreases in the prevalence
of infection in Latin America and Asia, estimated
prevalence rates for sub-Saharan Africa are equivalent to
those rst estimated by Stoll more than 60 years ago.
There have been few documented reductions in
hookworm prevalence in sub-Saharan Africa.
However, estimates of the number of people infected
do not translate into the disease burden caused by
hookworm, because not everyone infected will develop
the disease—morbidity is typically related to the intensity
of infection.
Today, the disease burden of most health
conditions is commonly assessed by the disability-
adjusted life year (DALY) metric.
For hookworm, DALYs
are determined mainly on the basis of disability weights
assigned to anaemia and cognition, with estimates
varying widely depending on the level of severity assigned
to each component. Therefore, there is a need to revise
estimates by combining data from attributable burdens
of anaemia,
childhood development,
and pregnancy-
related morbidities
resulting from hookworm infection.
Irrespective of which estimate is used, the greatest
disease burden caused by hookworm now occurs in sub-
Saharan Africa, and this is where the greatest challenge
for control lies.
Hookworm and anaemia
For the development of a vaccine against human hookworm
infection, the most critical endpoint is blood loss. Detailed
reviews of the biology and clinical features of hookworm
infection and disease are described elsewhere.
Intestinal blood loss is the major clinical manifestation
of human hookworm infection. Heavily and even
moderately infected patients with underlying iron or
protein nutritional defi ciencies can develop hookworm
disease—the clinical entity that specifi cally refers to the
resulting iron defi ciency and microcytic, hypochromic
anaemia (IDA) caused by hookworms feeding on
Hookworm-induced blood loss is estimated to
be as high as 9·0 mL of blood per day in heavy infections
(table 1), and hookworm burdens of 40–160 worms are
usually suffi cient to cause anaemia.
In school-aged
children and adults in resource-poor countries, where
host iron stores are often lower than those in developed
countries, there is a well-established intensity-related
relations between hookworm infection, intestinal blood
loss, and anaemia (table 2).
In children, chronic
hookworm disease is associated with physical growth
retardation and a correlation has also been seen between
hookworm burden and reduction in intelligence
quotient. There is increasing appreciation that
hookworm also exerts more subtle, yet profound,
adverse eff ects on childhood memory, reasoning ability,
and reading comprehension.
Most of these eff ects are
probably attributable to iron defi ciency. Hookworm
infection in children has been shown to adversely aff ect
school attendance, future productivity, and wage
earning potential.
In addition to their eff ects on children, WHO estimates
that because of increased physiological demands for iron
during pregnancy combined with malnutrition, more
than half of all pregnant women in developing countries
have IDA.
Severe IDA in pregnancy has been linked to
adverse maternal-fetal consequences including increased
maternal mortality, impaired lactation, infantile
prematurity, and low birthweight. An estimated 44 million
cases of hookworm occur during pregnancy worldwide,
with 7·5 million in sub-Saharan Africa alone.
Rationale for hookworm vaccine development
Unlike many other human helminthiases,
against hookworms does not protect against infection.
Indeed, the oldest people living in an endemic community
sometimes have the heaviest worm burdens.
an association was reported between levels of interleukin 5
and intensity of reinfection after anthelmintic treatment.
Anthelmintic drugs are highly eff ective at eliminating
existing hookworm infections, but they do not protect
against rapid reinfection,
leading to concerns about the
long-term sustainability of such practices.
A prophylactic
vaccine against hookworm infection is therefore a highly
desirable goal.
The development of such a vaccine, however, is a
formidable task. Helminths are multicellular pathogens
with large and complex genomes. They have an armoury
of defensive strategies that need to be overcome before
an eff ective vaccine can be deployed. Not the least of
these is their ability to skew the host’s immune response
Hookworm infection Eggs per gram of
n Mean blood loss
(SD) (mL/day)
Negative 0 50 1·24 (1·85)
Light 1–999 50 1·46 (1·07)
Moderate 1000–4999 50 2·96 (3·03)
Heavy ≥5000 50 8·79 (1·10)
SD=standard deviation.
Table 1: Amount of blood loss estimated by quantitative stool haem
analysis (Hemoquant) for Pemba Island children with diff erent
hookworm loads
Population group Country Attributable proportion (%) Reference
Infected Heavily infected
Pre-school children Kenya 14% 28% Brooker et al, 1999
Schoolchildren Tanzania 10% 31% Guyatt et al, 2001
Schoolchildren Zanzibar 24% 73% Stoltzfus et al, 1997
Schoolchildren and adults Brazil 22% .. Brooker et al (in press)
Pregnant women Kenya 10% .. Guyatt et al, 2000
Pregnant women Nepal 29% .. Stoltzfus et al, 1997
..=not reported.
Table 2: Anaemia attributable to hookworm infection
http://infection.thelancet.com Vol 6 November 2006
to favour their survival.
Hookworms secrete numerous
proteins with known and putative roles in
The immunomodulation induced
by hookworms and other helminths also has an
important (and in our opinion, mostly unrealised) eff ect
on the ability of people to respond to other
Feasibility of a hookworm vaccine—the
previous art
The rst evidence that hookworms could be attenuated
by ionising radiation was reported in 1959, when Dow
and colleagues
attenuated Uncinaria stenocephala larvae
with X-rays. Infective larvae of the canine hookworm
Ancylostoma caninum were later shown to be equally
suitable to attenuation by X-rays.
showed that single and double vaccination schedules
with a subcutaneous inoculation of 1000 X-ray irradiated
A caninum larvae could successfully protect vaccinated
pups against challenge infections with normal larvae.
Since A caninum infection can be established by either
oral or parenteral routes, Miller did a series of experiments
giving the irradiated larvae in a double vaccination
schedule by either oral or subcutaneous routes. Table 3
shows the results of experiments in which X-ray irradiated
and normal larvae were used as vaccines, and in which
oral and subcutaneous routes of vaccination were
Subcutaneous vaccination stimulated a more
uniform and higher level of resistance (determined by
numbers of adult worms reaching the intestine) than did
oral vaccination.
Miller postulated that the presence or absence of
migrating larvae in the lungs (somatic migration) was
the key factor in the greater effi cacy seen with the
irradiated larval vaccine when delivered subcutaneously
as opposed to orally.
This hypothesis was based on his
observation that three-quarters of the irradiated larvae
seemed to become “arrested before reaching the
intestine, with their demise probably occurring during
an extended sojourn in the lungs, a hypothesis that is
similar to the proposed mechanism of protection in
experimental irradiated schistosome vaccines.
also thought that protection against challenge infection
of vaccinated pups was attributable to three distinct but
interrelated factors that he referred to as the
characteristics of attenuation”: (1) reduction in the
infectivity of the larvae, measured by adult hookworm
establishment (worm burden); (2) reduction in the
pathogenicity of the resultant burden (host haemoglobin
levels); and (3) sterilising eff ect on female worm
fecundity as seen in reduced egg burdens (eggs per
gram of faeces). These criteria guided the successful
development and manufacture, and licensing in 1973
of a gamma-irradiated infective A caninum L3 vaccine
for canines.
This vaccine was eld tested by about
1500 veterinarians in continental USA, but manufacture
was discontinued in 1975. The overt reason for the rapid
discontinuation of the vaccine was poor sales, which
Miller blamed on the reluctance of veterinarians to
incorporate the vaccine into their routine vaccine
The discontinuation of the canine hookworm vaccine
has proved almost as instructive for researchers
developing a human hookworm vaccine as the
development of the irradiated larval vaccine itself. First,
a common complaint from owners and veterinarians
was the presence of eggs in the faeces after vaccination,
or as Miller put it the “unrealistic expectation for
successful banishing of all hookworm infection”.
human vaccines are sterilising in nature—ie, inducing
an immune response that kills the entire pathogen
load—which is essential for a vaccine against pathogens
that reproduce asexually, such as those responsible for
measles, poliomyelitis, and varicella. For the so-called
microparasites, vaccination is often sterilising, not only
reducing the incidence of disease in the vaccinees but
also indirectly protecting non-vaccinated, susceptible
individuals against infection, through a reduction in
transmission (herd immunity). A human hookworm
(or, indeed, any helminth) vaccine would be unlikely to
be sterilising in its eff ect. Instead, the major benefi t of
a human hookworm vaccine would be the reduction in
worm burden with a concomitant reduction in morbidity
(numbers 1 and 2 of Miller’s characteristics of
attenuation). The second reason for discontinuation of
the canine irradiated larval vaccine was, as Miller states,
“the failure of veterinarians to diff erentially diagnose
hookworm infection from hookworm disease”.
the pathology of hookworm is directly related to the
number of worms harboured by the host. As such, we
have gone to great lengths to explain that a human
hookworm vaccine would reduce the risk of heavy worm
burden and hence reduce host blood loss and its clinical
However, it was a minor and easily overlooked
problem with the canine irradiated larval vaccine that
proved the most informative for the development of a
recombinant hookworm vaccine—the effi cacy of the
vaccine depended on the viability of the larvae. In other
words, there was a requirement that the attenuated
larvae remain alive to secrete antigens upon host entry.
Design Protection % Lung exposure
Route Larvae Challenge Vaccination Challenge
Subcutaneous Irradiated Subcutaneous 88–93 (uniform) Entire and extended Entire
Subcutaneous Irradiated Oral 97 (uniform) Entire and extended Minimal
Subcutaneous Normal Subcutaneous 57 (variable) Entire and brief Entire
Oral Irradiated Oral 60 (variable) Minimal Minimal
Oral Irradiated Subcutaneous 84 (uniform) Minimal Entire
Oral Normal Oral 65 (variable) Minimal Minimal
Table 3: The previous art—comparison of subcutaneous and oral vaccination with irradiated and normal
Ancylostoma caninum L3 in dogs
http://infection.thelancet.com Vol 6 November 2006
This observation, together with earlier studies showing
the importance of larval-secreted antigens in mediating
protective immunity,
stimulated an antigen discovery
programme some 30 years later that set out to identify
the major secreted proteins of hookworm L3 as a target
for a human hookworm vaccine. Although irradiated
live parasites are not an acceptable proposition for
human vaccination, our laboratories have adopted
Miller’s characteristics of attenuation in canines as
some of the primary outcomes to be achieved by a
human hookworm vaccine—ie, reductions in worm
burden, pathogenicity, and fecundity.
A caninum infection resembles infection with the
human hookworms in a number of ways: (1) third-stage
infective larvae are acquired by their defi nitive host
through skin penetration (N americanus and A duodenale)
or oral ingestion (A duodenale), (2) larvae undergo a
short period of arrested development upon entry into
the host followed by a resumption of feeding and
development in host tissues, and (3) the number of
blood-feeding adult worms is directly related to
More importantly, larvae of both species
release antigens upon entry into the host that seem to
be tightly coupled to the developmental biology of the
parasite, especially to the transition to parasitism.
Miller’s early attempts to understand the mechanisms
underlying the protective eff ects of vaccination were
limited by the availability of immunological reagents for
dogs. However, even with these limitations, he accurately
determined that resistance was an immunological eff ect,
and not a mechanical one (larvae trapped in lung tissue),
by passively transferring protection through a
combination of serum and lymphoid cells.
advantage of the recent development of reagents for
canine immunology, Fujiwara and colleagues
that vaccination of dogs with irradiated larvae induced
antibodies that inhibited the penetration of infective
larvae through tissue in vitro, a possible mechanism for
the extended lung sojourn of irradiated larvae as
observed by Miller. Moreover, vaccination with irradiated
larvae induced high levels of antibodies and a strong
cellular response to crude larval antigen extracts. Raised
interleukin 4 production was also seen in vaccinated
dogs, especially in relation to interferon γ production,
implying that Th2 immune responses were involved in
generating protective immunity against hookworm
larvae. However, of most value for the development of a
recombinant vaccine was the observation that canines
vaccinated with irradiated larvae had strong antibody
responses to excretory/secretory products released by
hookworms upon entry into the host.
Feasibility of a hookworm vaccine—the state of
the art
Miller’s observations stimulated an antigen discovery
programme that set out to identify the major secreted
proteins of hookworm L3 with a focus on the canine
A caninum L3 secrete antigens in vitro into
culture medium only if they are stimulated in the
presence of glutathione (or its derivatives), or an
ultrafi ltrate of host serum, or both.
A caninum L3
stimulated in this manner were shown to release three
major proteins, including a metalloprotease involved in
tissue invasion,
and two cysteine-rich secretory
proteins termed ancylostoma secreted protein (ASP)-1
and ASP-2, which belong to the pathogenesis related
protein (PRP) superfamily.
PRPs are produced by
invertebrates, vertebrates, and even plants, and are so-
named because they are typically secreted in response to
pathogens and other stressors.
To assess the vaccine effi cacies of these (and at least
another 20) recombinant antigens, we formed the
Human Hookworm Vaccine Initiative (HHVI),
product development public–private partnership of the
Sabin Vaccine Institute in Washington, DC, USA. We
developed a scoring system that incorporated essential
criteria for determining an effi cacious human hookworm
vaccine (table 4). These include endpoints that focus on
Antigen Adult worm
Adult worm
(hamster) †‡
Reduced host
blood loss§
Eggs per gram
of faeces
Grading 0–5 0–5 0–4 0–4 0–2 0–3 0–2 ..
ASP-2 2 3 1 3 2 3 2 16/25 (64%)
APR-123332ND1 14/22 (64%)
CP-2 1 3 0 3 2 ND 1 9/22 (41%)
GST-1 2 3 0 1 2 ND 1 9/22 (41%)
ND=not determined. *Refl ects quintiles of reduction in worm burdens in dogs compared with controls. †Refl ects quintiles of reduction in worm burdens in hamsters
compared with controls. ‡Zhan B, Xiao S, Bethony J, Loukas A, Hotez P, unpublished data using N americanus and Ancylostoma ceylanicum in the hamster model. §Each grade
refl ects an increase of 0.5 dg/L haemoglobin above control group. ¶Refl ects tertiles of eggs per gram of faeces reduction compared with controls. ||Function or structure
known in hookworm (grade of 2) or in a related helminth (grade of 1), which enables biochemical assay development. **Association between antibody response and reduced
eggs per gram of faeces in people (number=strength of association). ††Protective homologues in other nematodes (grade of 2) or infectious agents (grade of 1). ‡‡Tally of
scores from each category.
Table 4: Ranking of candidate hookworm antigens on the basis of seven major criteria, and grading of each criterion to allow a fi nal score of vaccine
effi cacy to be tallied
For more information on the
Sabin Vaccine Institute see
http://infection.thelancet.com Vol 6 November 2006
pathology (blood loss, worm burdens), transmission
(faecal egg counts), ease of process development (known
function/structure of protein), and immunoepidemiology
(associations between immune responses and infection
intensities in naturally exposed/infected cohorts). The
ideal vaccine would reduce pathology to a minimum,
interrupt transmission, and be easy (and cheap) to
produce under current good manufacturing processes.
Once produced in soluble form, recombinant versions of
the major L3 excretory/secretory products (and
subsequently many other hookworm recombinant
proteins from the L3 and adult stages) were tested for
vaccine effi cacy in the canine and hamster models of
On the basis of this ranking system, recombinant
ASP-2 was selected as a lead vaccine candidate for
further process development, manufacture under
current good manufacturing processes, and clinical
testing. Among the studies pointing to the effi cacy of
ASP-2 was the immunological recognition of this
antigen by sera from dogs vaccinated with irradiated
Moreover, sera from dogs vaccinated either with
irradiated L3 of A caninum or adjuvant-formulated
recombinant ASP-2 inhibited larval migration in
and both groups of vaccinated dogs exhibited
signifi cant (p<0·05) reductions in host faecal egg counts
and adult hookworm burdens. By contrast, many of the
other major secreted antigens aff orded little to no
protection as recombinant vaccines in the canine
The animal protection experiments were also
extended to hamsters vaccinated with ASP-2 (Ay-ASP-2
cloned from Ancylostoma ceylanicum) and challenged
with A ceylanicum L3.
These studies also resulted in
signifi cant reductions in host worm burdens (p=0·025)
and faecal egg counts (p<0·001), as well as host blood
loss. Of interest is the observation that Hc24, an ASP-2
orthologue from Haemonchus contortus, is also a lead
candidate vaccine antigen.
Finally, in immuno-
epidemiological studies done in Brazil and China, there
was an association between the levels of IgE antibody to
ASP-2 and reduction in risk of acquiring heavy
hookworm burdens.
On the basis of the convergent data outlined above,
Na-asp-2 cDNA was cloned from the human hookworm
N americanus,
expressed in the yeast Pichia pastoris, and
antibody against the Na-ASP-2 recombinant protein was
shown to inhibit larval invasion in vitro
( gure 2). Initial
data from a phase I clinical trial in normal healthy
volunteers from the USA indicates that the vaccine is
immunogenic and well tolerated (Diemert D, Sabin
Vaccine Institute, Washington, DC, USA, personal
Development of a bivalent vaccine—targeting
As discussed earlier, it is generally accepted that a sterilising
helminth vaccine is unachievable, and moreover, a
sterilising vaccine is probably not essential for the
successful control of pathology and transmission of
helminth parasites.
An effi cacious hookworm vaccine
would therefore (1) reduce blood loss to a level below which
clinical anaemia develops, and (2) reduce egg output to a
level where transmission is interrupted and eradication
becomes a reality. We envisage a bivalent recombinant
human hookworm vaccine consisting of a protein that
targets (1) invasion and migration of the L3 (eg, Na-ASP-2)
and (2) blood-feeding by the adult hookworm (fi gure 3).
ASP-2 is expressed exclusively by L3.
A second antigen
that targets the blood-feeding adult parasite is therefore
α–ASP–2α–irL3 Control
Reduction in L3 penetration (%)
Figure 2: Percent reduction of A caninum L3 that penetrated skin in an in
vitro model of tissue migration by hookworm larvae
Each group of L3 was pre-incubated in sera from dogs that were vaccinated with
irradiated A caninum L3 (α-irL3), recombinant Ac-ASP-2 (α−ASP-2), or adjuvant
alone (control). Adapted from references 59 and 65.
Human hookworm vaccine (HHV)
Adult haemoglobinase
L3 antigen (ASP-2)
Inhibit haemoglobin
Reduced host blood loss
(hookworm disease)
1 Reduced worm burden
2 Reduced fecundity
1 Inhibit migration
2 Attenuate L3
Figure 3: Roadmap for the development of a bivalent human hookworm vaccine
ADCC=antibody-dependent cell-mediated cytoxicity.
http://infection.thelancet.com Vol 6 November 2006
essential so that the two major developmental stages
within the human host are under continuous
immunological assault in the vaccinated host.
Considering the vaccine effi cacy criteria set out in
table 4, the HHVI is currently focusing on molecules
used by adult hookworms during the blood-feeding
process. The hookworm ingests a portion of the
extravasated blood, and some of the ingested red blood
cells undergo lysis,
thereby releasing haemoglobin.
Haemoglobin, once released by lysis of ingested red cells,
is degraded with a suite of proteases, termed
haemoglobinases, in a semi-ordered pathway of
proteolysis in the intestine of the adult hookworm.
In view of the success of vaccinating sheep against
H contortus with haemoglobinase-rich gut extracts,
similar approach using recombinant haemoglobinases
from hookworms was proposed.
Vaccination of dogs
with the A caninum intestinal cysteine haemoglobinase
provided proof-of-concept that haemo-
globinolytic proteases are effi cacious vaccines against
hookworm infection,
resulting in a signifi cant decrease
in faecal egg counts (p=0·031) and stunting of adult
worms. The worms recovered from CP-2-vaccinated dogs
were shown by immunohistochemistry to ingest anti-CP-
2 antibody, which remained bound to the parasite gut at
necropsy. Furthermore, IgG from the vaccinated dogs
neutralised parasite protease activity in vitro.
Although vaccination with CP-2 established proof-of-
concept, the most promising preclinical data obtained in
canines so far has been with the cathepsin D-like aspartic
protease from A caninum, Ac-APR-1. Recombinant APR-1
from A caninum and its orthologue from N americanus,
Na-APR-1, cleaved host haemoglobin at the hinge region
(among many other sites),
a step that would facilitate
the unravelling of the haemoglobin tetramer and its
subsequent proteolysis by other aspartic proteases and
degradative enzymes. Vaccination of dogs with
recombinant Ac-APR-1 induced antibody and cellular
responses and resulted in signifi cantly reduced
hookworm burdens (p=0·065) and faecal egg counts
(p=0·018) in vaccinated dogs compared with control dogs
after challenge with infective larvae of A caninum.
importantly from a disease perspective, vaccinated dogs
were protected against blood loss and did not develop
anaemia (fi gure 4A). IgG from vaccinated animals
decreased the catalytic activity of the recombinant enzyme
in vitro, and antibody bound in situ to the intestines of
worms recovered from vaccinated dogs (fi gure 4B),
implying that the vaccine interferes with the parasite’s
ability to digest blood. In the coming years, a
haemoglobinase, like the Na-ASP-2 hookworm vaccine,
is anticipated to also undergo pilot manufacture under
current good manufacturing process guidelines, and
phase I clinical testing.
Roadmap for global access
The clinical testing of hookworm vaccines to the point of
proof-of-principle for their effi cacies would represent
major milestones. However, on the basis of the
experiences with the hepatitis B vaccine, there is concern
that following proof-of-principle it could take years or
even decades before a hookworm vaccine is made widely
available. Mahoney and Maynard
estimate 20–30 years
are often required before wide-scale availability is
achieved following proof-of-principle. To avoid long
delays in the distribution and deployment of hookworm
vaccines in areas of high transmission, the HHVI has
developed a global access roadmap.
22 42
Days post first vaccination
Haemoglobin (g/dL)
Figure 4: Haemoglobinase vaccines interrupt blood feeding
(A) The Ac-APR-1 haemoglobinase vaccine reduces anaemia in vaccinated but not control dogs after challenge
with infective hookworm larvae. Adapted from reference 89. LC=larval challenge. (B) Antibodies from dogs
vaccinated with recombinant Ac-APR-1 (frames 1 and 2) but not antibodies from control dogs (frames 3 and 4)
bind to and damage the microvillar surface of the gut when they are ingested during the parasite’s blood meal.
Reproduced from reference 89. in=intestine; mv=microvilli of intestinal cells; ro=reproductive organs.
http://infection.thelancet.com Vol 6 November 2006
The challenges for global access are formidable. As
one of the most prevalent infections of humankind, the
scale of the global hookworm problem is enormous. At
the same time, hookworm occurs predominantly in
rural areas, many of which are remote and often
inaccessible to health interventions. Compounding the
problem is that hookworm is one of 13 so-called
neglected diseases, which occur mainly in marginalised
and impoverished countries.
Such populations
represent the lowest priority markets for US and
European pharmaceutical manufacturers.
A nal
challenge is the possible requirement to establish a
health-delivery mechanism for administering a
hookworm vaccine. Overall, however, there is optimism
that these hurdles are surmountable. With that in mind,
the HHVI is developing a global access roadmap that
emphasises (1) a pivotal role for mathematical modelling
and cost-eff ectiveness studies, (2) unique global
partnerships for manufacture and distribution with
innovative developing countries and their vaccine
manufacturers, (3) pursuit of intellectual property, and
(4) establishing international consensus guidelines on
hookworm vaccine use.
Building international consensus: linking
vaccination with anthelmintic chemotherapy
We believe that a hookworm vaccine would be best
introduced by integrating it into ongoing WHO
anthelmintic chemotherapy (“deworming”) pro-
grammes in schools.
Linking these two control tools
would not only improve the outcome of vaccination but
also would decrease the requirement for frequent
dewormings and possibly delay the emergence of
anthelmintic drug resistance. In so doing, anthelmintic
vaccination would help with the sustainability of
deworming, now considered by some as something
Therefore, as a human hookworm vaccine
moves through the development pipeline, there is
enthusiasm for its integration into school-based
programmes developed under the auspices of
deworming, as well as greater school health initiatives
established by the World Bank and the Partnership for
Child Development.
Confl icts of interest
The authors are funded by HHVI, a product development public–private
partnership headquartered at the Sabin Vaccine Institute (SVI) in
Washington, DC, USA. AL and PJH are inventors on an international
patent application PCT/US02/33106 (fi led Nov 11, 2002), for a hookworm
vaccine. The patent was fi led in the USA, Brazil, India, China, and
Mexico. If awarded, the patent would belong to the George Washington
University with an exclusive license to the HHVI of the Albert B Sabin
Vaccine Institute, a non-profi t (501c3) organisation devoted to increasing
the use of vaccines worldwide. HHVI is funded mainly by the Bill and
Melinda Gates Foundation. The fi rst-generation hookworm vaccine, the
Na-ASP-2 hookworm vaccine was developed entirely in the non-profi t
sector through the HHVI-SVI. JB and SB declare that they have no
confl icts of interest.
The authors acknowledge the following funding bodies: the Bill and
Melinda Gates Foundation, the National Institutes of Health (NIAID and
Fogarty Center), the National Health and Medical Research Council of
Australia (NHMRC), and the Wellcome Trust. AL is supported by an
R D Wright Career Development Award from the NHMRC; JB is
supported by a fellowship from the Fogarty Center; SB is supported by a
Wellcome Trust Advanced Training Fellowship (073656).
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    • "However, the disease development by helminthic parasites usually depends on the intensity of infection [57] . Therefore, reducing the worm burden by vaccination , even not sterilizing, may significantly reduce the manifestation and seriousness of disease [57] . Nevertheless, new strategies are needed to improve the protection of vaccine against Trichinella infection. "
    [Show abstract] [Hide abstract] ABSTRACT: Trichinella spiralis infection induces protective immunity against re-infection in animal models. Identification of the antigens eliciting acquired immunity during infection is important for vaccine development against Trichinella infection and immunodiagnosis. The T. spiralis adult cDNA library was immunoscreened with sera from pigs experimentally infected with 20,000 infective T. spiralis larvae. Total 43 positive clones encoding for 28 proteins were identified; one of the immunodominant proteins was 20 kDa Ts-ES-1 secreted by Trichinella stichocytes and existing in the excretory/secretory (ES) products of T. spiralis adult and muscle larval worms. Ts-ES-1 contains 172 amino acids with a typical signal peptide in the first 20 amino acids. The expression of Ts-ES-1 was detected in both the adult and muscle larval stages at the mRNA and protein expression levels. Mice immunized with recombinant Ts-ES-1 (rTs-ES-1) formulated with ISA50v2 adjuvant exhibited a significant worm reduction in both the adult worm (27%) and muscle larvae burden (42.1%) after a challenge with T. spiralis compared to the adjuvant control group (p<0.01). The rTs-ES-1-induced protection was associated with a high level of specific anti-Ts-ES-1 IgG antibodies and a Th1/Th2 mixed immune response. The newly identified rTs-ES-1 is an immunodominant protein secreted by Trichinella stichocytes during natural infection and enables to the induction of partial protective immunity in vaccinated mice against Trichinella infection. Therefore, rTs-ES-1 is a potential candidate for vaccine development against trichinellosis.
    Full-text · Article · Aug 2015
    • "For personal use only. Vaccine [71]. For ascariasis and trichuriasis, the major criteria will include: level of protective immunity; amino acid homology between A. suum and A. lumbricoides or T. muris and T. trichiura; known or presumed mechanism of action; absence of prevaccination IgE among an endemic population; solubility and stability; and expression yield. "
    [Show abstract] [Hide abstract] ABSTRACT: Ascaris lumbricoides The Sabin Vaccine Institute Product Development Partnership is developing a Pan-anthelmintic vaccine that simultaneously targets the major soil-transmitted nematode infections, in other words, ascariasis, trichuriasis and hookworm infection. The approach builds off the current bivalent Human Hookworm Vaccine now in clinical development and would ultimately add both a larval Ascaris lumbricoides antigen and an adult-stage Trichuris trichiura antigen from the parasite stichosome. Each selected antigen would partially reproduce the protective immunity afforded by UV-attenuated Ascaris eggs and Trichuris stichosome extracts, respectively. Final antigen selection will apply a ranking system that includes the evaluation of expression yields and solubility, feasibility of process development and the absence of circulating antigen-specific IgE among populations living in helminth-endemic regions. Here we describe a five year roadmap for the antigen discovery, feasibility and antigen selection, which will ultimately lead to the scale-up expression, process development, manufacture, good laboratory practices toxicology and preclinical evaluation, ultimately leading to Phase 1 clinical testing.
    Full-text · Article · Jan 2014
    • "A product development partnership (PDP) was established in 2000 with an $18 million grant to the Sabin Vaccine Institute (SVI) to target N. americanus infections (Hotez et al., 2013). ASP-1 was among the first vaccine antigens tested, but performed poorly in challenge trials in the hookworm-permissive Ancylostoma ceylanicum–hamster model, so ASP-2 was advanced as the lead antigen for human trials despite having only marginally better protection (Goud et al., 2004; Bethony et al., 2005; Loukas et al., 2006). A Phase I trial in näıve volunteers in the United States, using N. americanus ASP-2 (Na-ASP-2) with the adjuvant Alhydrogel, was immunogenic and showed no serious safety concerns (Bethony et al., 2008). "
    [Show abstract] [Hide abstract] ABSTRACT: Abstract Soil transmitted helminths (STH) refer to several parasitic nematode species that infect over 1 billion people worldwide. Infections with Ascaris lumbricoides, Trichuris trichiura, and the hookworms Necator americanus and Ancylostoma duodenale cause significant morbidity in more than 450 million people, primarily children and pregnant women, resulting in over 39 million disability adjusted life years lost. Considerable effort and resources have been, and continue to be, spent on top-down, medical based programs to control STH infections, with little success. This review discusses the problems with these methods, and proposes a new emphasis on sustainable, long-term investments in sanitation-based approaches using improved latrines (the "box") to provide bottom-up, culturally appropriate and economically desirable solutions to STH control in endemic areas. One such approach is the use of biogas technology. Waste undergoes fermentation in specially designed septic systems, generating a methane gas mixture ("biogas") that can be burned to augment or replace household energy needs like cooking and light generation. Also, the effluent from the fermentation chamber provides a high-quality, nitrogen rich fertilizer. Using China as an example, the use of biogas technology as a solution to rural sanitation and energy problems is described, and its advantages over current strategies of mass drug administration and vaccination for STH control highlighted.
    Full-text · Article · Jan 2014
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