Download Transmission-Blocking Immunity against Malaria: From Antigen

Transmission-Blocking Immunity against Malaria: From Antigen Discovery to
Commercial Manufacturing
Running title: Transmission-Blocking Immunity
Abstract
Lack of an effective vaccine, parasite resistance to anti-malarial drugs, and resistance to
insecticides of the anopheline mosquito vectors have caused enormous mortality and morbidity
due to malaria worldwide. Now, while genetic manipulation or chemical incapacitation of the
vector is faced with serious ethical and technical problems, production of transmission-blocking
vaccines (TBVs) seems to be effective. TBVs block parasite sexual stages within the mosquito
vector. Several TBVs candidate molecules have already been identified. Through recent
magnificent studies, two candidates passed to phase1 of human trial. In spite of the successes
achieved in the potential application of TBVs against malaria, many formidable barriers have
remained unsolved regarding commercial manufacturing of TBVs. In this review, all the above
mentioned issues are discussed in detail and some debouchments are presented with respect to
previous studies.
Keywords: Transmission-blocking vaccines, Anopheles, Plasmodium, Antigens
1. Introduction
More than half a million people die of malaria each year, 90 per cent of them children under 5. 1
Along with the death rate, morbidity left after the disease is responsible for major economic
losses basically in under-developed countries. Malaria causes almost 50 million disability
adjusted life years 2a signal that indicates current strategies are not efficient and there is a need
for other tools if to reduce the spread of malaria. Plasmodium falciparum, one of the most lethal
of the malaria-causing species, keeps threatening humans, especially pregnant women and
children, in many areas around the world.
-3
Members of the Anopheles gambiae complex
include seven closely related species (An. gambiae, An. arabiensis, An. melas, An. merus, An.
1
bwambae, and An. quadriannulatus A and B), from which An. gambiae, An. stephensi, and An.
arabiensis are the most important vectors of human malaria.4
Classic strategies for malaria control have to be supported by new methods Genetic manipulation
of mosquitoes is a new promising strategy in this respect. Although we can easily produce
transgenic mosquitoes today,5-6 replacement of natural vector populations with genetically
manipulated populations needs improvement of robust genetic approaches.7 Plasmodium takes
various forms in the vertebrate host and invertebrate vector which need to be understood if to
develop a new strategy for malaria control.7-8
After the ingestion, the gametocytes differentiate into gametes, fertilization occurs, and the
zygote develops into an ookinete, that forms oocysts in the midgut epithelium. When the oocysts
rupture, the sporozoites from the haemolymph invade the salivary glands to be injected into a
new host. The transition between the gametocytes and ookinetes, between the ookinetes and
mature oocysts, and the development from the midgut sporozoites into salivary glands
sporozoites are considered as weak links in the sporogonic development during which the
parasite is extremely vulnerable.9 Malaria parasite has to pass completely different physiological
barriers including the peritrophic matrix, epithelia of the midgut, and salivary gland. Therefore,
current transmission-blocking approaches target Plasmodium inside the mosquito midgut and
block the invasion of the mosquito to the midgut and salivary gland epithelia. In this review,
TBVs will be thoroughly discussed while a focus will be put on pathways, strategies, weaknesses
and strengths, history, candidate antigens, and future of TBVs. The review is an attempt to
discuss the improvements made in the development of a vaccines that blocks the transmission of
malaria within a community. The review gives a detailed account of the TBV candidates.
2. Limitations of potential malaria vaccines strategies
2
Genetic engineering of attenuated malaria parasites for vaccination is a promising strategy;
however, poor understanding of the genetic manipulation of Plasmodium along with lack of
enough genomic and biological information have posed serious challenges to the method.
Another strategy is to neutralize sporozoites as they enter the blood stream.
10
This kind of
vaccine has the potential of completely preventing infection, but the enormously complex
epidemiology of malaria; the five species – each of innumerable strains; and the vast genetic,
geographical, social, and developmental diversity of human race conspire to create a huge
spectrum of exposure and a very large number of different host–parasite relationships.11 Other
strategies have their limitations too. In this condition, transmission- blocking vaccines are more
promising. In most malaria-endemic locations, even a partial TBV coverage would reduce the
morbidity rate of malaria. In high endemic areas, the use of TBVs together with traditional
methods like ITNs and IRS could stop malaria transmission completely. 12
3. TBVs: Definition and History
The concept of malaria TBV technique was developed in the 1950s, when Huff and colleagues
mentioned transmission-blocking immunity could be induced in chickens vaccinated with a
combination of avian malaria asexual and sexual parasites.13 In the course of the next thirty
years, using monoclonal antibodies and surface labeling of gametes and zygotes, the major
candidate antigens, have been recognized in P. falciparum sexual stages.14 The TBVs block
pathogen development inside the vector and subsequent transmissions to a non-infected
vertebrate host. Transmission-blocking vaccines in malaria are expected to suppress the
3
developmental stages of the malaria parasites within the mosquito midgut. The first 48 h after the
ingestion of an infective blood meal is essentially the most critical time period for the parasite
development inside the mosquito.15 In this period, the Plasmodium fertilization happens and the
subsequent zygotes develop into ookinetes. The development from gametocytes to salivary gland
sporozoites is considered as a weak link in sporogonic development.16 Blagborough et al. (2003)
provided direct evidence that selected transmission-blocking intervention inhibits transmission
from the vertebrate to the insect by 32% and reduces the basic reproduction number of the
parasite by 20%. So, the use of transmission-blocking interventions alone can eliminate
Plasmodium from a vertebrate population.17 The idea of TBVs can be seen in figure 1.
4. Molecules for TBVs
TBVs candidate antigens are molecules derived from the pathogen or the vector that reduce the
pathogen transmission from the infected to the uninfected hosts. These candidates may be monoor polyclonal antibodies from the vertebrate host or recombinant DNA plasmids containing the
gene encoding such molecules.17 TBV candidates should have these qualities: first of all they
should be inducing antibodies that prevent the development of Plasmodium in the mosquito
vector; secondly, restricted antigenic diversity is necessary for TBV antigens; furthermore,
adjuvants should be capable to induce the output of excessive antibody titers after just one
injection from the vaccinated individual as no natural boosting will take place. Molecules from
the parasite or mosquito could have such properties. Typically, adjuvants are used along with
antigens to enhance the immune response of the vertebrate. Adjuvants enhance immunity to
antigens by a range of mechanisms, which have not yet been clearly understood .18-22 The
4
particular antibodies produced against the pathogen or vector antigens will prevent the pathogen
development within the vector, following a blood meal on a vaccinated and infected individual.
5. Use of TBVs
The basic equation used in the estimation of TBVs affectivity is as follows 23:
RoTBV = RoI (1 – c)
where Ro is the basal reproductive quantity outlined as the number of the new cases of malaria
estimated to be seen in a non-immune human individual from a particular untreated case of
malaria in a non-immune person. Transmission is not sustainable below the critical value of
Ro=1. So, RoTBV is a Ro smaller than a specific TBV coverage, Ro I is the preliminary Ro in a
population prior to the TBV deployment, and c is the proportion of the community with an
efficient TBV vaccine protection. Malaria transmission ultimately ceases when RoTBV falls
below 1,. The expected coverage of a target community using an efficient TBV will be 50% to
90%.24 The extent of protection achievable in a given scenario would rely on the logistics
affecting the target community. Stability of human population with little immigration of
unvaccinated people into the vaccinated community is necessary for an effective TBV
vaccination. Malaria transmission may be very focal due to the localized nature of the breeding
sources of the mosquito vectors and their restricted dispersal range – typically from a hundred
meters to one or two kilometers –. Therefore, the particular TBV-vaccinated individual reduces
malaria inoculation rates to the members of his or her family. In case of TBV for regional
elimination of malaria, it could possibly be expected that under low endemic transmission
circumstances, a TBV may eradicate malaria inside a locality. However, in moderate endemic
areas, TBV may transfer malaria transmission rates near the range that malaria is possible to be
eliminated by other interventions.
5
Additionally, malaria transmission can be reduced in low endemic regions by TBVs. In low
endemic conditions, reducing malaria inoculation rates using a TBV could be obviously helpful,
because it might reduce the occurrence of malaria in proportion to the efficient TBV coverage. In
most extreme endemic regions, the consequences of reducing malaria inoculation rates via TBV
would probably be a total reduction in disease and mortality. This might move the chance of
death from malaria to older individuals resulting from a delay to the achievement of protective
immunity. The TBV-induced immunity might, even at comparatively low coverage, considerably
retard the emergence of a malaria epidemic. So, TBVs may fully abort a potential epidemic of
malaria or prevent it from reaching a higher level. Lastly, it could possibly be mentioned that a
TBV, when deployed together with different sorts of malaria vaccine or anti-malarial medicine,
could be efficient in stopping the escape and spread of the mutants resistant to these vaccines or
drugs. This could apply significantly to asexual blood stage vaccines and anti-malarial drugs to
which the possibilities of resistant mutants arising can be high.
6. Strengths and weaknesses of TBVs
Development of an efficient TBV raises a number of challenges. As mentioned before,
transmission reduction in medium to high zones seems beneficial. In low zones, however, data
are lacking but there is an obvious risk to enter an unstable situation with higher risks. TBV has
been considered altruistic because no direct personal protection is obtained. This may be true
from a biological perspective but not from a public health viewpoint. TBV will probably be the
most effective in areas where the preliminary basic reproductive rate of malaria (Ro) is low. 3,4,8,9
6
A TBV has the advantage of blocking the spread of the escape mutants that are resistant to
asexual-stage vaccine components or to anti-malarial medications. Hardly could candidate TBV
antigens be compared to the pre-erythrocytic- and erythrocytic-stage vaccine because they
usually are not under selection pressure within the human host
25
(figure 1). One significant
advantage of TBV is that there are many target molecules or antigens that can be utilized as
TBVs. Much of the work conducted on TBV development has centered on parasite antigens
expressed in the mosquito midgut, while transmission blocking of malaria can also be achieved
by focusing on the mosquito antigens necessary for a successful development of the parasite in
its vector 26 (table 1).
7
Figure 1 (Source : authors) Different malaria vaccines: in the pre-erythrocytic stage, sporozoites
get into a liver and antibodies used to block them. Merozoites digestion of red blood cells will
happen in the blood stage. The sexual stages of parasite development and transition from
ookinete to oocyst will happen in the mosquito stage and this is the best time to apply the
transmission blocking vaccines (TBVs).
8
Mosquito-based antigens do not encounter the second host immunity; consequently, TBV would
not be boosted by the natural malaria infection. Here, the specific adjuvant capable of boosting
the immunogenicity of the TBV candidate is required.27 TBVs will reduce the mosquito
fecundity and survivorship.28 TBVs might completely abort an epidemic of malaria or prevent it
from reaching a high level. TBV may also be counted as an excellent substitute to the problems
(expense and environmental) related to insecticides application.
Table 1: Different types of malaria vaccines
Type of vaccine
Goals of vaccination
A. Before emergence from liver
disease will be prevented
Reduce disease by a vaccine
Pre-erythrocytic
that combines partially
effective pre-erythrocytic
and blood stage components
Reduce disease by reducing blood
Blood Stage
stage asexual parasite burden
Mosquito Stage
A. Eradicate
B. Limit spread of parasites
resistant to vaccines
C. Prevent epidemics in areas of
unstable malaria
transmission
Target population and situation for
vaccination
A. Travelers and residents in low
transmission areas (e.g. Iran)
B. Children and pregnant women
in areas of high transmission
(e.g. Africa)
Children and pregnant women in areas
of high transmission (e.g. Africa)
A. Entire community in isolated
areas of low transmission
B. In any situation and in
combination with blood stage or
pre-erythrocytic vaccines, entire
population before high transmission season
7. Antigen-Based TBVs
These antigens are expressed on the surface of sexual stage of Plasmodium. Antigen-Based
TBVs includes Plasmodium falciparum proteins Pfs25, Pfs28, Pfs48/45, and Pfs230 as well as
their orthologs in Plasmodium vivax in addition to Pys21 and Pys25.
Pys21 and Pys25
9
Two monoclonal antibodies, Pys21 and Pys25 (against 21-kDa and 28-kDa ookinete surface
proteins), from P. yoelii block completely P. yoelii infectivity to An. Stephensi.
29
Anti-Pys25
monoclonal antibody has a complete transmission-blocking activity and its bioactivity is more
potent than anti-Pys21 MAb.
30
Pys21 and Pys25 interfere with the development of zygotes to
ookinetes in the absence of complement and antibody-dependent cell-mediated cytotoxicity.30
Pfs25
Pfs25 is a 25kDa protein produced on the surface of zygote and ookinete phases of P. falciparum
and consists of four tandem epidermal development factor (EGF) subunit.31 After the fertilization
of the female gamete by the male gamete, Pfs25 is expressed in the mosquito.32 There is no
evidence that a single TBV will probably be efficient against all Plasmodium species.33 A
number of intrinsic impasses related to TBVs, together with bad immunogenicity of Pfs25 and
the possibility for polymorphism amongst Plasmodium populations in totally different areas have
been addressed lately. To deal with these problems, different prime-enhance methods have been
followed and significantly extra immunogenic formulations have been provided.34-36 Be that as it
may, the problem of polymorphism or clonal variability amongst distinct P. falciparum or P.
vivax strains is still a palpable issue. In contrast to Pvs25, Pfs25 is less polymorphic.37 The
primary hurdles to this sexual TBVs is the inherent need for large quantities of antibody to fully
suppress parasite growth within the mosquito.17Infectivity of An. freeborni to P. falciparum was
once reduced to nearly 40% by using virus delivery system as 25 µg/mL of Pfs25 rabbit
produced monoclonal antibodies was supplied to the blood meal. Saul (2008) efficacy model for
mosquito stage transmission blocking vaccines for malaria predicts that the current formulations
of Pfs25 are likely to achieve useful reductions in the transmission when tested in human field
trials.38
10
The requirement for prime-antibody titers might impose a restriction on the utility of the present
parasite-based TBV formulations. The most well-known adjutants having been used for Pfs25
boosting include Freund’s and muramyl tripeptide (MTP-MF59). A phase I clinical trial of the
Pfs25 antigen using Montanide ISA 51 adjuvant was completed.39 Although anti-Pfs25 human
serum inhibited P. falciparum oocyst in An. stephensi by more than 90%, reactogenicity (native
and systemic) in human volunteers prevented Montanide ISA 51 for being used as an adjuvant
with Pfs25.39 DNA-based Pfs25 vaccines use plasmids in expressing this antigen. The most
effective transmission blocking activity was observed by injection of plasmids encoding Pfs25
(infectivity reduced up to 96.2-96.6%).40 This is quite different for some other antigens. For
example, TBV25H secreted by Saccharomyces cerevisiae is the recombinant type of the
molecules tested in the human phase I clinical trials (D. C. Kaslow et al., unpublished data).
Vaccination of mammals with TBV25H using aluminum hydroxide adjuvant shows a complete
transmission-blocking activity.41
Pfs28
Pfs28 zygote surface proteins of P. falciparum (and their equivalents for P. vivax) are presented
after the gametogenesis inside the mosquito midgut and are usually not present in the
gametocytes, as they circulate into the blood. As a result, they will most likely not be boosted
after the malarial infection. Therefore, a vaccinated community could possibly be prone to the
risk of an epidemic return of malaria as soon as their vaccine-induced transmission-blocking
immunity has diminished. New formulations should thus be developed to increase the efficient
lifetime of TBV-induced immunity. Since Pfs28 is barely expressed within the midgut and never
in the vertebrate host, these antigens have not been under the selection pressure by the host
immune system, and the antigenic variation of Pfs28 seems to be more restricted than most
11
vaccine candidates existing in the pre-erythrocytic and asexual blood stages.31 It has been
proven that vaccination with each P. vivax and P. falciparum antigens induces full transmissionblocking in the model systems.42
Pfs48/45
Targeted gene interfere evaluations have proven that Pfs48/45 has a crucial function in male
gametogenesis, a very important aspect of the sexual reproduction success of the parasite. 43 A
strong positive correlation between anti-Pfs48/45 antibody and transmission blocking bioactivity
has been observed in malaria endemic regions, so Pfs48/45 is regarded as a major candidate for
the TBV development.44 Nonetheless, the efforts to provide full length recombinant Pfs48/45 in
a functional conformation have largely remained unsuccessful. Additional development of a
transmission blocking vaccine based on Pfs48/45 is supported by the observation that the
purified protein exhibited longer-lasting functional immunogenicity in baboons.45 Even 5-6
months after the final booster immunization, the antibodies elicited through vaccination
continued to successfully block infectivity (not only in oocyst burden but also a proportion of
infected mosquitoes) of P. falciparum gametocytes in the vectors.46 It has been hypothesized that
a vaccine triggering the response might be extra boosted through natural infection and therefore
help in keeping higher antibody levels in the vaccinated community.47 Correctly folded Pfs48/45
protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in mice.48
Lately, Chowdhury et. al.46 (2009) have produced a full length antibody in correct folding and
after a single immunization, they achieved a blocking affectivity of higher than 93% in MFA.46
Additionally, it has been realized that Pfs48/45 exhibits significantly better potency in An.
stephensi.
49
In most malaria-endemic locations inside Africa, Pfs48/45 coverage, would reduce
disease and death due to malaria. In many situations of low malaria endemicity in africa,
12
transmission could be stopped by Pfs48/45 TBVs. Pfs48/45 is by far the most advanced EUdeveloped malaria TB vaccine candidate. PF10C is a subunit of Pfs48/45 that has been produced
as R0-PF10C. Immunization with 100% properly folded R0-PF10C induced a transmissionblocking activity in 100% of the immunized mice. Some project objectives are rapidly preparing
for clinical TBMV testing in Africa.50
Pfs230
Pfs230 was presented on the plasma surface of gametocytes when they developed in the human
host. Pfs230 expressed as soon as Plasmodium developed to stage V gametocytes within the
human host.51 One study observed a positive correlation between the ability of the serum to
immunoprecipitate Pfs230 and block P. falciparum transmission in a mosquito feed assay.52
Various studies have shown that Pfs230 might contribute to immune evasion by eliminating the
immunodominant amino terminus earlier than the gamete is released and is available in direct
contact with the serum. Recombinants encoding Pfs230 have neither been very immunogenic in
mice, even following covalent attachment of tetanus toxoid, nor produced transmission-blocking
antibodies.53
WARP
Von Willebrand factor A domain-related protein (WARP) can be observed in mature ookinetes
and the following oocysts.54 WARP might cause ookinetes to bound to the vector midgut
epithelium and accelerate the differentiation of ookinete to oocystbesides the interaction with the
vector basal lamina. Ookinete to oocyst transition was decreased considerably when vectors fed
on an infected animal that was previously immunized using the anti-WARP polyclonal
13
antibody.55 This means that the antibody interferes with the WARP function by spotting the
protein on the floor of the Plasmodium and makes it a target antigen for a TBV.55 The antiWARP-produced polyclonal antibody effectively inhibits up to 92% of the parasite development
within the vector.56 The excessive similarity (61%) between the PvWARP and PfWARP residues
at the amino acid level 54 indicates a huge conservation of the WARP basic arrangement between
these two particular Plasmodium species. Theoretically, and based mostly on these discoveries, it
is suggested that WARP be utilized as a common TBV against combined P. vivax and P.
falciparum. Nonetheless, it must be considered that it is not a clear antibody-binding site for
WARP, that plays a role in the transition from ookinetes to oocyst in the vector midgut, are
positioned inside the similar amino acid areas of each species.54
Other pathogen molecule-based TBV candidates
Pfg27/25 on the surface of gametocyte has been studied for transmission-blocking immunity.57
This antigen is usually not exposed on the surface of the Plasmodium although it develops within
the vector, making it an uncommon TBV antigen. Pvs25, a Pfs25 ortholog, is presented on the
surfaces of both zygotes and late ookinetes, while Pvs28 is expressed on mature ookinetes.58
Using Alhydrogel® (aluminium hydroxide gel) as the adjuvant, phase 1 human clinical trial with
Pvs25 was carried out in the U.S.59 and no reactogenicity (side effects) was observed. The
rPvs25/IFA immunization constantly conferred a complete blockade, but the local reactogenicity
was significantly greater than the Alum-adjuvanted or adenovirus-vectored immunization
routine.60
8. TBVs by molecules from insect host
14
These antigens were presented inside insect vector tissues. Moreover, not only do these
antibodies blocked the development of P. falciparum and P. vivax in numerous Anopheles
species, but also may be used as vector-blocking vaccines, because they reduce the vector
survivorship and fecundity.61
FBN9
FBN9 is one of the fibrinogen domain immunolectin gene families with a minimum of 61 genes
within the An. gambiae genome.62-64 The FBNs pattern recognition receptors enable them to
attach to parasites and play an important function in the mosquito's natural immunity, along with
the physiological processes related to blood feeding.65 FBN9 was up-regulated when An.
gambiae was invaded by P. falciparum ookinetes; however, no up-regulation was observed in
responses to P. berghei ookinetes invasion.66 An. gambiae mosquito’s susceptibility to both P.
falciparum and P. berghei infections will increase by FBN9 knockdown in RNAi gene silencing
assays.66 According to the study, Garver et al. have shown that manipulation of the mosquito's
immune system can produce a near-complete loss of the vector's ability to transmit an unusually
virulent strain of the human malaria parasite. Also they proved that FBN9 is indeed a part of the
Caspar silencing–mediated anti-Plasmodium mechanism.66
APN
When inactively transferred to P. falciparum infected, mice polyclonal antibodies against the Nterminal portion of Aminopeptidase N (APN), could considerably reduce the quantity of
oocysts in. An. stephensi.67 It is not clear when APN plays a role. Nor have its interactions not
been mapped to a specific parasite molecule. APN does not immediately interfere with any
15
Plasmodium molecule, and the antibody-mediated suppression is a result of an indirect
mechanism. 68
Carboxypeptidases B
Two Carboxypeptidases B, including CPBAg1 and CPBAg2, are expressed in the midgut of
Anopheles.69 The ingestion gametocytes of P. falciparum up-regulate the expression of both
cbpAg1 and cpbAg2, suggesting that CPB is involved in the P. falciparum development and
could be a candidate molecules for a TBV. Parasite development inside the An. gambiae is
blocked by using polyclonal antibody against CPBAg1, and the number of the infected vectors is
reduced by more than 92%.70 CPBAg1 is completely presented inside the mosquito midgut and
is not exposed to the selective pressure in humans. Mammal immunization experiments have
shown that a single injection can lead to excessive antibody titers and transmission-blocking
immunity.71Furthermore, CPBAg1 monoclonal antibody reduces mosquito reproductive
functionality, an impact which will enhance the influence of the vaccination by decreasing the
native vector inhabitants.71
MG96
A membrane-bound midgut protein called MG96 has been incompletely characterized in An.
gambiae and An. stephensi recently.72 This protein is expressed on the midgut epithelium not
only in blood-fed vectors, but also in unfed mosquitoes, indicating that the expression of MG96
is constituent and it should not be induced due to blood feeding. MG96 displays a dosedependent blocking impact on the Plasmodium yoelii development in An. Stephensi. In one
experiment, the parasite development inside the mosquito midgut was blocked by 100%. MG96
16
identifies midgut antigens which can be particularly positioned alongside the comb border which
lines the apical side of the gut epithelial cells the lumen.72
Saglin and SGS
P. gallinaceum sporozoites attack were inhibited by the antibody against the An. aegypti salivary
glands.73 In an in vivo bioassay, the MAb against the 100-kDa protein inhibited Plasmodium
yoelii sporozoite attack of salivary glands and there was a trend of inhibition.74 This revealed that
An. gambiae salivary gland proteins are available to MAb that block the sporozoite invasion of
the salivary glands. It has been shown that two salivary gland antigens, aaSGS1 and saglin,
perform as the binding sites for the Plasmodium sporozoites and can be used as the TBV target.75
The sporozoite invasion of the salivary glands is related to the Saglin antigen as well as some
others.75 The use of the salivary gland proteins for TBVs would circumvent the ‘lack of
boosting’ drawback confronted by the mosquito-midgut-based TBVs.76 A brief description of all
candidate antigens along with their explanation can be found in Table 2 (a, b).
9. Adjuvants
The action of the antigen/adjuvant formulation is a result of multiple factors. Therefore, the
immune reaction obtained with a known mixture of an antigen and adjuvant is vaccine-specific.
Currently no information can be found that might enable an extrapolation to a different antigen
or even to a similar formulation presented by another distinct path. Thus, the particular
individual antigen/adjuvant vaccine formulation is what is presently licensed in the U.S. There is
no regulatory guideline that would be valid to every case. The use of adjuvants to enhance the
TBV antigen response is a critical problem to be resolved, because particular kinds of adjuvants
17
induce reactogenicity both in humans and in animal models.77-78 Salts aluminum known as Alum
is considered to be a weak adjuvant for antibody induction in humans but is generally applied in
vaccines in clinical use.79 It has been observed that Salts aluminum primarily provides a Th2
reaction in mammals.80 Montanide ISA720, a kind of water-in-oil emulsion adjuvant, has been
applied with the malaria vaccine antigens to increase the speed and duration of the vaccinespecific protective response in animal experiments and in human trials.81 Oligonucleotides
containing CpG motifs that increase the total antibody titer or functional titers have proven
effective adjuvants for DNA- or protein-based and have been used for human trials; they
82
primarily elicit a Th1 response in mice. Pvs25 and Montanide ISA51 induced local and systemic
adverse effects in rhesus monkeys
39
, whereas Pvs25-H Montanide ISA 720 immunization
induced 10 fold more antibody in comparison to the aluminium hydroxide gel combination, and
also resulted in native reactogenicity.38 In an experiment, the monkeys immunized with lower
doses of Pfs25/Montanide ISA 720 did not show any reactogenicity.38 Reactogenic reactions due
to Pfs25/Montanide ISA 720 might be the result of a qualitative difference in the immune
response between the first and second boosting. This might be prevented if the second dose is
applied quite a few months later with a smaller dose.38 Table 3 shows the different adjuvants
used in the TBVs experiments.
Table 2a: Different antigens candidate for Plasmodium transmission-blocking vaccines
Table 2b: Different antigens candidate for Mosquito transmission-blocking vaccines
18
Type
Antige
n
Mechanism
Strength
Pvs25
(39, 59.
83,84)
interfering with
transformation of
zygotes to
ookinetes
The ability to induce both cell-mediated and humoral
immunities as well as a long-lasting memory T cell
response
Antibody-dependent cell-mediated cytotoxicity was not
observed
Pfs25
(39,83)
blocking oocyst
development in
mosquitoes
Not expressed in the vertebrate host and less likely to
be subject to naturally occurring immune selection
pressure
inhibiting oocyst
formation
PTB
V
Pfs28
(85)
Vaccination with both the P. vivax and P. falciparum
antigens induces complete transmission-blocking in
model systems
Monoclonal antibodies to both antigens block
transmission in membrane feeds
It is not under selection pressure by the host
Pfs48/4
5 (46,
48, 50)
causing
deficiency in
zygote formation
Monoclonal antibodies to the 48/45 completely block
transmission in membrane feeds.
Expressed on the gametocyte, and so making boosting
of antibody response a possibility.
Pfs230
(51, 52,
53,)
contributing to
immune evasion
by removing the
immunodominant
amino terminus
Monoclonal antibodies to the 230 completely block
transmission in MFA
Expressed on the gametocyte, and so making boosting
of antibody response a possibility
WARP
(54, 86)
inhibiting Oocyst
formation
A clear mechanism of antiparasite activity
Nearly complete block transmission
19
Weakness
Devel
The inhibitory effect was
just observed in therodent
malaria model and there
are serious doubts that it
could be observed in
human ones
Poor immunogenicity
The only forms of Pfs25
shown to be effective have
been those expressed
in vaccinia virus or in yeast
A phase I t
antigen usin
ISA 51 ad
carri
Potential for polymorphism
among parasite populations
in different geographical
sites
vaccinated community
could be at the risk of an
epidemic return of malaria
Yeast expr
high yields
and
Correctly fo
by ind
biologically
Folding problem makes it
difficult to express in
different vectors
Due to large size of this
molecule it is not clear
which part is responsible
for transmission blocking
activity
Not expressed in the
vertebrate host and so
not subject to natural
boosting
Phase 1 hu
trial was
Expressed
without
confo
Producing
antibody
Fragment
expressed as
fusion
It has been
E.coli rec. p
evidence o
Type
Antigen
Mechanism
Strength
FBN9 (6266)
blocking oocyst
formation
Conserved gene family
role in the mosquito's innate
immunity
Ability to act against diverse
Anopheles species
APN (67,68)
not clearly understood
but probably indirect
mechanism (e.g.
blocking access to an
adjacent molecule on the
midgut that is the real
ligand for the parasite)
MTBV
No toxicity at in vivo studies
anti-APN antibodies are more
effective in blocking parasite
invasion than anti-CPB antibodies
20
Weakness
Not subject to natural
boosting
A method for estimating the
concentration of antibody
used in membrane-feeding
assays varies among
investigators
FBN9 ha
innat
immune
Casper
Full
amp
Clon
Bind
antibo
CPB (69-71)
inhibiting CPB activity
that in turn reduced the
available resources in
arginine and lysine
below the
threshold level required
for parasite development
MG96 (72)
disrupting the ookineteto-oocyst transition
Saglin &
SGS (73-76)
inhibiting sporozoite
invasion of salivary
glands
Expressed exclusively in the
mosquito midgut and is not
exposed to selective pressure in
humans
single injection can induce high
antibody titers
CPBAg1 antiserum reduces
mosquito reproductive capacity
Low concentrations of this MAb
can still effectively block parasite
development in the mosquito
midgut
Boosting a problem will not be a
problem anymore using Saglin &
SGS
Anti-CPB can result in
selection of resistant
mosquitoes due to decrease
reproductive fitness of the
vector
MG96 exhibits a dosedependent blocking effect
that limit its application
Appropriate adjuvant
formulations or
employment of the delivery
systems seem to be
extremely important
in inducing an antiparasite
immune response
Table 3: Different adjuvants used in Transmission-blocking vaccines s
Adjuvant
Antigen
Vaccinated
animal
Targeted
pathogen
Vector
Alum
Pvs28
Mouse
Freund (FCA)
Pfs45/48
Mouse
Alum
Pvs25
Mouse
P.
falciparum
P.
falciparum
P. vivax
Alum
Pvs25
Mouse
P. vivax
Montanide ISA
720
Alhydrogel®
Montanide ISA
720
Alum
Pvs25
Monkey
P. vivax
Pvs25
Pvs25
Human
Monkey
P. vivax
Pvs25
Monkey
P. vivax
Montanide ISA
720
Cholera toxin
Pfs25
Human
Pfs25
Mouse
Freund (FCA)
WARP
Rabbit
P.
falciparum
P.
falciparum
P. vivax
An.
stephensi
An.
stephensi
An.
stephensi
An.
freeborni
An.
albimanus
An.
freeborni
An.
freeborni
An.
stephensi
An. dirus
Freund (FCA)
CPBAg1
Mouse
P. berghei
21
An.
stephensi
An.
gambiae
Reference
87
48
56
19
34
59
88
88
41
36
36
69
cpb
have
MFA
Cloning
succes
M
Recen
confirm
that are
saliva an
puta
10.
TBVs manufacturing future
Malaria is the disease of poor countries. These involve areas which can neither purchase nor
produce TBVs. So, the question remains as to whether malaria will ever be eliminated? The
answer is a NO. Today’s strategies for malaria control obliviously suffer from the lack of a
comprehensive insight including ethical, commercial, and scientific details. Moreover, no single
liable organization has shouldered the responsibility to organize various aspects of the problem.
So problems we are facing here to produce TBVs are beyond the WHO capabilities. The most
important aspects regarding the TBVs commercial production to be taken into consideration
involve R&D, technical support, liability, and manufacturing (figure 2). Liability is related to the
World Health Organization (WHO) and governmental and non-governmental health
organizations (NGOs). The most important problem goes backs to the lack of workable liquidity
(fund), and regarding technical support, the lack of commitment among different organizations
for making promising attempts to manufacture TBVs. Having solved the above problem, we face
even a more serious problem of the stock product drugs which will be left unpurchased. Who
will purchase TBVs? African countries are not rich enough. Thus once again we get back to the
liability and commitment of the WHO to guarantee TBVs production. These four areas are
interrelated and success in one leads to progress in the others and vice versa. Table 4 shows the
details of these four areas in TBV production.
22
Figure 2: Different aspects of commercial production of transmission blocking vaccines (TBVs)
and their related specified problems.
Table 4: Different challenges in TBVs commercial production.
23
Problem area
Problem definition
potential formulations at or forthcoming a grade
appropriate for trying out as human TBVs are not
accessible now for many of candidate antigens
probably TBVs not boosted by malarial infection
achieving optimum levels of coverage
Technical
binding affinities of the antibodies have never been
determined
the method for estimating the concentration of
antibody used in membrane-feeding assays varies
among investigators
assessment of transmission-blocking antibodies
entails membrane feeding assays are often
unreliable
lack of strong R&D
lack of commercial interest
Market
preference of liver- and bloodstage vaccines rather than TBVs
Problem solving
Develop low unit cost vaccines
Apply the proteins that present on the
gametocyte through the infection
within the human host (such as
pf230); so making possible boosting
of transmission-blocking antibody
reaction throughout an infection
Develop compatibility and even
synergistic in combination
with other malaria vaccine
components
Conduct more experiments
Conduct more uniform experiments
Do in vivo assessment
Provide budget for targeted R&D
milestone via the use of the epidemic
countries' facilities, researchers,
scientists, and corporations inside
regions similar to Iran, China,
Pakistan, and African countries
Outline added value for TBV which
includes instant and nonstop saving
in therapy costs $2 billion globally in
1995), Long run expenditure ($350
million out of Africa in 1995) and
$300 million save in development
costs per medicine or vaccine
Misplaced to resistance
WHO will be in charge to ensure
TBV is purchased in the target areas
inside African counting given the
risks, expenditures, and liabilities
Find a way that TBVs can be used for
travelers and the military entering
malaria-endemic regions
the concept that a TBV system requires the
vaccination of people who is not going to
themselves get advantages has usually been raised
as an ethical opposition to TBVs
Make usage of TBV mandatory
lack of track record for purchasing and providing
global access to current vaccines
Develop a good vaccine delivery
systems involving registration,
documentation, and report.
24
11. Concluding remarks
Transmission-blocking vaccines opened a new window to the world of malaria. These types of
TBVs need more support to be commercially produced. To date, some potent antigens like
FBN9, APN, CPB, MG96, Saglin & SGS, Pys21, Pfs28, Pfs48/45, Pfs230, and WARP with high
transmission blocking activity have been recognized and their mechanism of action have been
partly or completely explored. Moreover, the approval of the P. vivax Pvs25 antigen as TBV
during a phase I human trial was a significant achievement. Assigning enough funds to TBVs
research and developments in addition to precise planning for future research will make TBVs
available in market. As researchers in the area of TBVs, we should indicate that the parasitebased TBV vaccine development faces lots of obstacles linked to the incredibly efficient
immune-evasion approaches evolved by Plasmodium and to the complicated conformational
structure of many Parasite antigens which impair the successful large production of recombinant
TBV antigens. There are lots of skepticism and a few uncertainties concerning the realistic value
of TBV as an approach for restricting the burden of malaria. The authors believe that malaria
transmission rates have to be reduced to zero to eliminate malaria from a selected environment.
So, the use of the traditional malaria preventing strategies and vaccines together with regular
medicine would by no means be useful in aiming this goal. TBVs will reduce the emergence and
spread of the parasites resistant to different malaria vaccine elements and even to anti-malarial
drugs. In this respect, the use of TBVs might significantly extend the efficient lifetime of other
malaria vaccine components and probably also of anti-malarial drugs. In case of TBVs, the
possibilities for the resistant mutants emerging in comparison to asexual blood stage malaria
vaccines, liver stage vaccines, and anti-malarial drugs will be the least. It is because of this fact
that sexual stages do not multiply and are prepared in comparatively low numbers, when they are
25
under TBV-induced immune pressure. There may be little industrial interest in a transmission
blocking vaccine whose relevance is to poor nations, where malaria is widespread. Thus, while a
transmission-blocking vaccine would be of large community benefit, it lacks industrial support
and requires a house within the public sector that may champion its development.
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List of abbreviations:
World Health Organization: (WHO)
Governmental and non-governmental health organizations: (NGOs)
Malaria transmission blocking vaccine(s) : MTBVs
Transmission blocking vaccine(s): TBVs
Anopheles: An
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