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*Corresponding author: E-mail: shadrackkimenju@gmail.com; 
 
 
 

Asian Journal of Immunology 
 
4(1): 77-84, 2021; Article no.AJI.69854 
 

 
 

 

 

Advances and Challenges in Transmission-Blocking 
Malaria Vaccine Development. A Systematic Review 

 
Laura Nyawira Wangai1, Shadrack Kimenju Kahiro2*, Kenny Kimani Kamau1, 
David Waweru Nderu1, Immaculate Marwa Nyaiseba1, David Butto Amarch1, 

Mark Kilongosi Webale1 and Elly Munde1 

 
1
School of Health Sciences, Kirinyaga University, Kutus, Kenya. 

2
Department of Biochemistry, Jomo Kenyatta University of Agriculture and Technology, P.O. Box 

62000-00200, Nairobi, Kenya. 
 

Authors’ contributions  
 

This work was carried out in collaboration among all authors. All authors read and approved the final 
manuscript. 

 
Article Information 

 
Editor(s): 

(1) Dr. Cynthia Aracely Alvizo Báez, Autonomous University of Nuevo Leon, Mexico. 
Reviewers: 

(1) G-Halli R. Rajasekariah, Biofirm Pty Ltd, Australia.  
(2) Wagner Quintilio, Instituto Butantan, Brazil. 

Complete Peer review History: https://www.sdiarticle4.com/review-history/69854 

 
 
 
 

Received 15 May 2021  
Accepted 19 July 2021 

Published 03 August 2021 

 
 

ABSTRACT 
 

Malaria continues to cause enormous human suffering throughout most of the tropics and 
subtropics. In sub-Saharan Africa alone, it is estimated that about two million children die each year 
due to malaria. Vector control and malaria chemotherapy strategies that were previously effective 
in controlling and treating malaria, respectively, are now largely ineffective owing to the spread of 
insecticide-resistant mosquitoes and drug-resistant parasites. A vaccine targeting the sexual stages 
of the parasite and block transmission is needed to reinforce current malaria control and 
eradication efforts. Here, we review the status of malaria transmission-blocking vaccines. We 
focused on the efficacy, progress and development challenges of transmission-blocking vaccines. 
Pfs25 and Pfs48/45 are essentially the lead-candidates malaria transmission-blocking vaccine and 
should be studied further in clinical trials. Our review highlights the need to develop novel malaria 
elimination interventions, particularly an effective malaria vaccine with transmission-blocking 
activity.  
 

Review Article 



 
 
 
 

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78 

 

Keywords: Malaria mosquitoes Pfs25 Pfs48/45 vaccines; drug-resistant parasites plasmodium. 
 

1. BACKGROUND INFORMATION 
 

Malaria is caused by unicellular Plasmodium 
protozoan parasites that are injected by female 
Anopheles mosquitoes. P. falciparum, which 
causes the most severe form of malaria, is a 
substantial threat today, causing more than 200 
million clinical cases and 409,000 deaths in 2019 
[1]. While the level of malaria has dropped over 
the last 10 years, this decline has recently 
stalled, with some regions experiencing a 
resurgence in the number of malaria cases [2]. 
Unfortunately, resistance to artemisinin-based 
combination therapy (ACT), the current frontline 
treatment for malaria, is present in Southeast 
Asia and now spreading in Africa [3]. This 
highlights the need for novel strategies in malaria 
prophylaxis and treatment, especially an effective 
vaccine that can induce a protective antimalaria 
immune response. Even though the development 
of a malaria vaccine is still a challenging task, the 
development of several vaccine candidates is 
ongoing.  
 

Malaria vaccines can thus be broadly 
categorised into three groups based on the 
parasite stage being targeted, namely 
sporozoite, liver-stage and asexual blood-stage 
vaccines. Liver-stage and asexual blood-stage 
malaria vaccines (a transmission-blocking 
vaccine), unlike an anti-sporozoite vaccine, do 
not either directly protect a vaccinee against 
malaria infection, reduce parasitaemia or prevent 
disease; but rather employs all these four 
strategies. Anti-sporozoite vaccines would help 
by preventing malaria-infected travellers from 
transferring malaria parasite into a malaria-free 
zone [4]. The malaria parasite is particularly 
vulnerable to immune intervention during the 
sexual and sporogony stages because (1) 
parasites pass through a substantial numerical 
bottleneck-with about 100 parasites of the billions 
of asexual blood-stage parasites developing into 
gametocytes, get ingested by mosquitoes and 
subsequently developing into oocysts; (2) the 
sexual and sporogony stages persist 
extracellularly for hours unlike other parasite 
stages; (3) many of the candidate antigens tend 
to be highly immunogenic owing to their low 
antigenic diversity as a result of no immune 
selection in host since parasites do not express 
vaccine candidate antigens while in the human 
host  [5].  
 

Although antibodies targeting gametocytes 
(sexual stages) are not able to block human 

infection or disease, they can potentially stop 
malaria transmission. Recently, there has been 
increasing interest in the use of gametocyte-
specific antigens as candidates for a 
transmission-blocking vaccine (TBV) [6]. Such a 
vaccine would induce antibody production that is 
taken up alongside the gametocytes during a 
mosquito blood meal and neutralize the parasites 
in the mosquito midgut upon expression of the 
antigens. Key sexual-stage antigens that have 
been considered as candidate TBV include 
Pfs48/45, Pfs230, Pfs25, and Pfs47 [7,8]. It has 
been reported that antibodies from malaria-
exposed individuals or vaccinated animals 
against these antigens could block malaria 
parasite development in mosquitoes [9]. These 
findings offer an impetus to test human 
monoclonal antibodies against TBV antigens, 
such as those generated recently against Pfs25, 
for the ability to block malaria transmission. 
 

2. ROLE OF TRANSMISSION-BLOCKING 
VACCINE 

 
The main goal of transmission-blocking vaccine 
development programs is to eradicate malaria 
parasites using an inexpensively produced, 
stably formulated, and easily administered 
vaccine. It is unlikely that a single antigen subunit 
vaccine will be enough. It noteworthy however 
that transmission-blocking vaccines will have a 
great impact on malaria control when they are 
combined with other vaccines or control 
modalities. These may include bed nets, 
protective vaccines, and massive chemotherapy 
administration, in controlling or even perhaps 
eradicating malaria in geographically isolated 
areas e.g. islands or isolated human and 
mosquito populations) [10]. In instances where 
malaria is eradicated, long-term control will be 
necessary to prevent the re-introduction of the 
parasite. This could be accomplished by 
enforcement of mandatory TBV vaccination for 
persons returning from areas where malaria 
persists and/or demonstrate evidence of prior 
vaccination with TBV.  

 
The adoption of TBV combined with other 
malaria control strategies may boost the 
effectiveness of existing malaria control efforts. 
Firstly, a combination of TBV and multistage 
malaria vaccines could contribute substantially in 
curtailing the spread of’ mutant parasites that 
may be resistant against the protective 
components of the vaccines by reducing malaria 



 
 
 
 

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79 

 

transmission to a rate that would make an 
otherwise partially effective protective component 
highly effective [11]. Secondly, co-administration 
of TBV with effective anti-malarial drugs will help 
prolong malaria drug efficacy and delay the 
emergence or spread of drug-resistant parasites. 
Finally, TBV could reduce morbidity/mortality 
caused by the spread of the virulent strains of the 
parasite. Previous studies have demonstrated 
that the more an individual is exposed to new 
strains of parasites, the more likely they are to 
develop severe clinical disease [12–14]. 
However, it is still unclear whether this 
phenomenon is caused by a new “virulent” 
parasite strain that is biologically different (e.g., 
has a different cytoadherence profile) from other 
strains circulating in a geographical area [13] or it 
is simply caused by an immunologically new 
strain to the host. 
 

3. CANDIDATE VACCINES 
 
Prior fertilization, extracellular gamete (male and 
female)-specific antigens are the earliest point 
during sexual development to which an antibody-
mediated transmission-blocking effect has been 
described. The primary function of gametes is to 
find and fuse with a gamete of the opposite sex, 
fertilization, in a process that occurs within 
minutes after a mosquito ingests the sexual 
stages [15]. Since the components of the 
complement cascade are present in the blood 
meal are still active for minutes to hours after 
ingestion, the parasite has developed protective 
mechanisms against complement-mediated lysis 
[16]. It is therefore not surprising that the two 
major mechanisms implicated in antibody-
mediated transmission blockade are directed 
towards gamete surface proteins that interfere 
with fertilization and sensitization of gametes to 
complement-mediated lysis [17]. Other 
mechanisms that contribute to transmission 
blockage may involve a cellular component, such 
as antibody-dependent cell cytotoxicity (ADCC) 
and opsonization have not yet been described, 
but may occur as well.  
 
The major advantage of transmission-blocking 
vaccines that target pre-fertilization antigens is 
that immune responses are boosted by each 
subsequent natural infection. Whether boost will 
increase the titer of transmission-blocking 
antibodies or extend the longevity of 
transmission-blocking immunity is yet to be 
determined. Furthermore, antigen 
immunogenicity also needs to be studied since 
seroepidemiological studies indicate that less 

than 50% of humans carrying gametocytes 
contain detectable antibodies to either of the two 
lead pre-fertilization vaccine candidates, namely 
Pfs230 and Pfs48/45 [17]. Other pre-fertilization 
vaccine candidates are Pfs2400, Pfs40 and 
Pfg27/25 [18]. 
 

3.1 Immune Response To Pfs25 
 
Malaria vaccines that target both human and 
mosquito infections have the potential to               
impact malaria control. The importance of 
developing such a vaccine is underscored in a 
WHO World Malaria Report, which indicates 
stagnation in the progress towards reducing 
global malaria cases since 2015 [19]. The 
malaria vaccine RTS, S, which targets human 
infections as indicated by its impact on clinical 
disease, has completed phase 3 testing and is in 
pilot implementation studies in three African 
countries, namely Kenya, Ghana and Malawi 
[20,21]. Efforts to develop a vaccine that disrupts 
mosquito infection, known as a malaria 
transmission-blocking vaccine (TBV), have been 
ongoing since the reporting of induced TBV-
induced immunity in chickens against 
Plasmodium gallinaceum in 1976 [22]. The pace 
of malaria TBV development, until recently, has 
been hindered by the lack of capacity to               
produce candidate antigens for clinical testing 
[22].  
 
Currently, clinical testing is are limited to                
Pfs25, a P. falciparum-specific antigen. Pfs25 is 
a 25 kDa sexual-stage protein present on the P. 
falciparum zygote and ookinete surface in the 
mosquito midgut. The leading Pfs25 TBV is a 
chemically conjugated vaccine comprising of a 
Pichia pastoris expressed Pfs25 and the                 
carrier protein ExoProtein A (EPA) or a 
recombinant detoxified form of Pseudomonas 
aeruginosa ExoToxin A9. The Pfs25-EPA 
conjugate has the biophysical characteristics of a 
nanoparticle with a size similar to the hepatitis B 
virus-like-particle used in RTS, S [23]. ex vivo 
standard membrane feeding assays                    
(SMFA) during phase 1 trials of Pfs25-EPA 
conjugates formulated with Alhydrogel™, an 
aluminium-based adjuvant conducted in the 
United States of America, and Mali revealed that 
four doses are required to generate antibody 
titers that significantly reduce parasite 
transmission  [22]. However, poor clinical trial 
results have halted the development of Pfs25-
EPA as a stand-alone TBV. Which can              
probably be tackled by using the correct carrier 
protein. 



 
 
 
 

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3.2 Immune Response To Pfs230 and 
Pfs48/45 

 
Another family of sexual-stage proteins with 
cysteine-rich domains includes the antigens 
Pfs230 and Pfs48/45 that have been targeted for 
TBV development. However, until recently,              
there was no recombinant antigen preparation 
available with the identity, purity, and                      
quality necessary for human clinical trials [24].           
Of the two, Pfs230 cysteine-rich domain was the 
first antigen to be produced. It consists of a 230 
kDa sexual-stage protein that is composed of 
fourteen 6-cysteine-rich domains that suitable for 
human clinical testing [25]. Recombinant               
Pfs230 domain 1 (Pfs230D1M) is well-
characterized and has shown to induce 
transmission-blocking antibodies in small animals 
using the SMFA16 [24]. Parasite-derived                 
antigen (Pfs23016A) has been shown to induce 
the production of transmission-blocking 
monoclonal antibodies (4F12). A phase 1 safety 
and immunogenicity study evaluating Pfs230 D1-
EPA nanoparticles formulated on Alhydrogel™ 
has been completed [24]. Pfs230D1M is 
conjugated chemically to EPA forming 
nanoparticles with a similar size as hepatitis B 
virus10.  
 

3.3 Post Fertilization Target Antigens  
 

Several post-fertilization target antigens have 
been described, including Pfs28 (D&y& Kaslow. 
submitted), Pfs25. chitinase and mosquito midgut 
late trypsin [26,27]. The two major mechanisms 
mediating blockade post-fertilization involve 
arresting the morphological transformation of the 
round, sedentary zygote to the oblong, motile 
ookinete and preventing the egress of the 
ookinete from the blood meal to the midgut 
epithelium basal lamina [11]. Preliminary 
evidence suggests that transmission-blocking 
antibodies may also interfere with normal 
sporozoite development. However, the exact 
mechanism(s) by which this occurs is not yet 
known though there is increased gametocyte 
clearance and impaired development in later 
stages [28]. The major advantages of post-
fertilization target antigens are immunogenicity 
and limited pre-existing antigenic diversity, 
unequivocally reflecting the lack of prior immune 
selection on these antigens. Since these target 
antigens are expressed late in sexual sporogonic 
development, high antibody titers may be 
required to account for antibody degradation 
from proteolysis in the blood meal and boosting 
after a natural infection may not occur.  

4. FIRST GENERATION TRANSMISSION-
BLOCKING VACCINE, Tbv25h 

 
One of the most advanced candidate 
transmission-blocking vaccine in development is 
a 6 histidine-tagged protein TBV25H 
(Transmission-Blocking Vaccine based on Pfs25 
with a Histidine tag) secreted from recombinant 
Succharomvces cerevisiae [29]. A modified fed-
batch fermentation protocol was developed that 
optimizes recombinant protein production using 
the simplest and least expensive fermentors 
available. The 6 histidine-tag is used to purify the 
protein with ease from the yeast culture 
supernatant by affinity chromatography with 
nickel-NTA agarose. The protein is further 
purified by simple gel filtration chromatography 
and then adsorbed to alum. Thus, in keeping with 
the goal of technology transfer to newly 
industrialized countries, a simple fermentation 
and post-fermentation process was developed 
that should allow the ultimate end-users to 
produce the vaccine themselves [30]. However, 
TBV25H/alum is not an ideal transmission-
blocking vaccine for three reasons; it may require 
refrigeration, in vivo studies have demonstrated 
that multiple injections may be required to induce 
the production of transmission-blocking 
antibodies and immunity is relatively short-lived. 
In its current formulation, TBV25H will be 
invaluable tool for determining the safety of 
TBV25H/alum and help improve our 
understanding on how antibodies work to block 
infectivity in vitro and how it can be elicited in 
humans. A series of human clinical trials will be 
required [5]. 
 
Assessing the efficacy and the impact of a TBV 
poses some unique challenges [11]. The lack of 
reliable in vitro correlates of protective efficacy 
has limited the early phases of protective malaria 
vaccine development; the latter field-testing 
phases of protective vaccine development are 
straightforward. In contrast, the early phases of 
development of a TBV have the advantage of a 
reliable (but tedious) in vitro assay for assessing 
transmission-blocking activity, the membrane 
feeding assay in which in vitro cultured 
gametocytes are mixed with test serum and fed 
through an artificial membrane to starved 
mosquitoes. The degree to which the test serum 
inhibits infectivity is determined 1 week after an 
infectious blood meal by scoring mercurochrome-
stained mosquito midguts for oocysts [31]. 
Nevertheless, in vivo efficacy for transmission-
blocking vaccines will require labour-intensive, 
expensive field testing [21].  



 
 
 
 

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The assessment of the actual efficacy of a TBV 
requires field testing unlike any vaccine tested to 
date, malaria or otherwise. Although a Phase I 
trial designed to establish safety and 
immunogenicity has been used to assess 
efficacy in vitro (using the membrane feeding 
assay) and a Phase IIb (naturally acquired 
parasite infection rather than experimental 
parasite challenge) can be used to assess in vivo 
efficacy by feeding laboratory-reared mosquitoes 
directly on infected volunteers, designing and 
executing robust Phase III trials (i.e. testing 
efficacy in the field) that determines the impact of 
the vaccine on natural transmission and on 
preventing morbidity and mortality is difficult [8]. 
This is because, in Phase III trials, the statistical 
or study unit is not individual-based but requires 
a well-defined and, at least partially, isolated 
population of sufficient size in a malaria-endemic 
or seasonal malaria area. The setting must be 
such that the rate of transmission is not so high 
that a possible positive effect is missed, but not 
so low that the observed effect would have little 
consequence as a public health measure. The 
control and test study units, or communities, 
must have little interchange between vaccinated 
and unvaccinated populations but must be 
similar to allow comparative analyses between 
among sites [22]. Depending on the rate of 
transmission, the endpoints that are chosen, the 
similarity in malaria transmission and endemicity, 
the existence of a malaria transmission control 
programme (e.g., the presence of P. vivax 
transmission), and the cooperation of all 
individuals in each community, as few as one or 
two test villages and an equal or greater number 
of control villages may be all that is necessary to 
preliminarily evaluate efficacy [28].  
 
Several ethical issues, however, must be 
adequately addressed before undertaking these 
human clinical trials. Despite the enormity of the 
health problems that malaria poses worldwide. 
an effective malaria vaccine has eluded us. Lack 
of understanding as to what the mechanism of 
protective immunity is, limited research funding, 
and perhaps lack of the appropriate technologies 
necessary to develop an effective vaccine has 
probably contributed to the delayed development 
of effective malaria vaccines. Novel approaches 
and technologies may be required such as TBVs.  
 

4.1 Sequence Polymorphisms of Vaccine 
Candidates 

 

Considering that amino acid sequence 
polymorphisms present a plausible bottleneck for 

malaria subunit vaccine development [32], there 
is a major concern that B-cell epitopes targeted 
by transmission antibodies might be highly 
variable [13]. To assess whether any of the 
polymorphisms affect the antibody affinity 
towards Pfs48/45, serum samples from rats 
immunized with R0.10C were tested in the SMFA 
with three distinct P. falciparum isolates of Asian 
and African origin, i.e. NF54, NF166 and NF135 
[33]. Taken together, available data from 
sequencing and antibody binding studies of field 
isolates suggest that parasite diversity is unlikely 
to become an obstacle for the clinical 
development of Pfs48/45-based vaccines [13].  
 
Expression of a recombinant vaccine has been 
hindered by the inability to produce sufficiently 
high yield recombinant proteins that refolds into 
the native structure required for the induction of 
TBV antibody production [34]. Proper folding of 
many cysteine-rich proteins, including Pfs48/45, 
depends on the correct formation of disulphide 
bridges [35]. In eukaryotes, the oxidizing 
environment of the endoplasmic reticulum (ER) 
provides a milieu for disulphide bonds formation, 
a process that is mediated by ER oxidoreductase 
1 (Ero1) and protein disulfide isomerase (PDI) 
[36]. P. falciparum possesses four protein 
disulfide isomerases (PfPDIs) of which, PfPDI-8 
and PfPDI-11 have been expressed in 
gametocytes [37]. Pfs48/45 and Pfs25 have been 
expressed in different eukaryotic expression 
systems possessing Ero1 and PDI analogous but 
some systems have a track record for production 
of recombinant malaria antigens the reported 
yields of properly folded recombinant Pfs48/45 
have been disappointingly low [38,39]. 
 

4.2 Preclinical Studies  
 
Numerous animal studies have shown that 
Pfs48/45, Pfs25 and other candidates 
vaccination elicits antibodies with the capacity to 
inhibit parasite fertilization in mosquitoes as 
determined in the SMFA [28]. In general, a 
recombinant protein which assumes a proper 
fold, as determined by the reactivity with mAbs 
against conformational epitopes, elicits high 
levels of transmission-blocking antibodies [21]. In 
contrast, miss-folded protein does not elicit 
transmission-blocking antibodies suggesting that 
correct folding of critical epitopes involved in 
transmission-blocking immunity is essential for 
an efficacious TBV [21]. Adjuvants are vital for an 
efficacious subunit-vaccine by enhancing 
seroconversion rates and concentrations of 
functional antibodies [40]. In the preclinical 



 
 
 
 

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adjuvant selection, it is important to focus on 
those with a human clinical development path 
with records of both safety and manufacturability 
[28]. Whether this level of vaccine-specific 
antibodies is comparable to naturally occurring 
antibodies against Pfs48/45 is unknown. This is 
mainly because most studies of naturally 
acquired immunity have reported antibody data 
as categorical variables (either positive or 
negative) rather than continuous variable. The 
availability of new recombinant proteins will help 
future studies to estimate the level of vaccine-
specific as well as naturally acquired antibodies. 
The ability of newly designed adjuvants to 
enhance levels of functional antibodies against 
Pfs48/45 has been investigated in small rodents 
using recombinant proteins produced in the L. 
lactis expression system [6]. Formulations 
containing the synthetic TLR4 agonist 
glucopyranosyl lipid adjuvant (GLA) or a 
combination of synthetic lipid adjuvant (SLA) and 
saponin containing QS21 in a liposome 
formulation (SLA-LSQ) induce the highest titers 
of antibodies to sexual stage antigens. Both the 
GLA and SLA agonists have been assessed in 
human clinical trials as a part of vaccines against 
several infectious diseases suggesting they 
might be useful for the development of a 
TBV[35].  
 

Viral vectors are considered an attractive 
alternative to protein-based vaccines because 
they circumvent the need for production and 
purification of properly folded protein and 
because of their ability to effectively induce both 
humoral and cell-mediated immune responses 
[9]. In recent years this approach has been 
explored extensively [41]. For instance, Pfs48/45 
has been tested in a ChAd63-MVA heterologous 
prime-boost regime as a glycosylated and non-
glycosylated recombinant protein [42]. Mouse 
IgG against non-glycosylated Pfs48/45 shows 
transmission-blocking activity in SMFA using 
homologous NF54 parasites and to a lesser 
extent also in the direct membrane feeding assay 
(DMFA) using P. falciparum parasites collected 
from naturally exposed gametocyte donors [6]. 
Collectively, these findings suggest that Pfs25 
and Pfs48/45-based subunit vaccines have the 
capacity to elicit functional antibodies in humans. 
 

5. CONCLUSION 
 

This review highlights the need to develop novel 
malaria elimination interventions in particular an 
effective malaria vaccine with transmission-
blocking activity. Pfs25 and Pfs48/45 are 

essential lead-candidates for a transmission-
blocking vaccine and should be explored further 
in clinical trials. 
 

CONSENT 
 
It is not applicable. 

 
ETHICAL APPROVAL 
 
It is not applicable. 
 

COMPETING INTERESTS 
 
Authors have declared that no competing 
interests exist. 
 

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