Challenges for obtaining more effective malaria vaccines depend on precise selection of antigenic motifs and understanding the complexity of Plasmodium spp. life cycle. Naturally expressed antigens are characterized for being weak immunogenic when tested as vaccine components, thus these have to be strategically modified to render them immunogenic. A molecular clue in this pursuit is provided by the chemical peptide-bond processing by peptidases, which follows a multistep pathway including ephemeral high energy molecular complexes known as transition states. Thus, we have proposed non-natural peptide-bond isosteres as transition states mimetics, and therefore, stabilizing these high-energy states with site-directed designed immuno-mimetics have demonstrated being a rational approach for stimulating antibody populations harboring multiple functional capacities. Therefore, peptide-bond substitutes constitute a coherent pathway towards obtaining selected immuno-active compounds from specific plasmodial molecular objectives. Chemical strategies for synthesizing peptido-mimetics and antimalarial selected trials lead us to assess a number of peptide-bond substitutes for obtaining immuno-active and structurally defined molecules. Plasmodium antigens expressed on merozoite, sporozoite and gametocyte stages have been selected as targets and subsequently modified based on the presence of either a high-binding motif or a potential HLA-reading frame. This new family of immuno-mimetics is and efficient neutralizing antibody inducers when tested in in vitro and in vivo experiments, thus representing a new generation of malaria vaccine components.
- synthetic vaccine
- non-natural elements
- functional antibody
Malaria is spreading from old
A number of difficulties inherent to many causes among the pathogen resistance to antimalarials, poor coverage of public health programs and natural human host genetic restrictions make the finding of an effective malaria vaccine be an urgent need.
Malaria vaccine development has constituted a big challenge for researchers, up-to-date about 236 vaccine candidate prototypes are being tested. Approaches based on strategies such as immunization with irradiated sporozoites of
Natural immunity to malaria is related to hemoglobin structure, some disorders such as thalassemia confer resistance to
Our group has introduced in this pursuit some non-natural elements to be incorporated into synthetic antigens with the aim of governing both antigen presentation as well as specific B-cells for functional neutralizing antibody stimulation. Some of these non-natural elements included capture sequences for stimulating antigen degradation and others for the peptide-bond structure modulation. Peptide-bond isosteres included reversal configuration thereof, urea motifs and reduced amide as peptide-bond surrogates all constituting a novel immunogen family herein named as immuno-mimetics.
Once strategically incorporated into selected antigens, produced peptide-bond surrogates overcome non-desirable properties of native non-modified antigens such as cytotoxicity and hemolytic profiles, besides prolonging these new molecules half-life and a remarkably strong immuno-stimulating activity that can be associated to the newly introduced freedom degrees to the 3D structure of immuno-mimetics.
Also, we have consolidated the female BALB/c animal model for malaria vaccine candidate testing based on controlled challenging performed to immunized animals with two rodent malaria strains, being those
2. Global health statistics, economical and environment determinants
In 2015, time for fulfillment of the millennium development goals (MDGs) was getting closer to the end, and a consequent protocol comprising 17 sustainable development goals (SDGs) constitute the next step. In its annual report, World Health Organization (WHO) analyzed 15 years of advances of those proposed MDG and evaluated the next challenges for the coming years.
As reported in world health statistics 2015 issued by the WHO , undernutrition was the main cause of mortality in an assessed 45% of all deaths of children under 5 years of age. In the 1990–2013 period, the estimate of underweight children in third-world countries decreased from 28 to 17%, and a sustainable decreasing rate to 16% was expected for the end of 2015. In spite of those proposed efforts for achievement of MDG, these numbers are not still sufficient to the goals. The proportion of underweight children declined globally from 25% in 1990 to 15% in 2013.
Worryingly, poverty is strongly associated with public health especially to problems related to high transmission of infectious diseases. As observed in Figure 1A, among the main causes of deaths among children under 5 years (in neonatal and post-neonatal ages), between 2000 and 2013 are responsibility of infectious diseases, pneumonia, malaria, HIV/AIDS, measles, diarrhea and sepsis are the main reasons of mortality accounting 13 and 35%, respectively. Malaria represents 7% of children mortality mainly in the post-neonatal period between 1 and 59 months of age.
In the last year, 836 million of the world population lived on less than US$1.25 daily in comparison with 1.9 billion in 1990. In those the so-named poor countries, 14% of the people lived on less than US$1.25 daily in the same year, regarding the 47% in 1990. Getting closer to an amount of US$2 daily has been difficult at higher poverty levels.
The most inhabited counties of the world such as People’s Republic of China and India have been crucial for world reduction in poverty (indeed India remains the earth’s country having most extreme poverty; Figure 1B) such reduction can be associated to growth of central economic sectors and labors. Other factors such as income transferring, remittances and evolving new demographic profiles have had a lesser impact. However, those efforts have not been enough since one of each seven people in poor countries live on less than US$1.25 daily. In the sub-Saharan countries, more than 40% people are living in extreme poverty in 2015. In the countries having middle-incomes, the 73% of the Earth’s poverty is found .
Figure 1B displays the top 10 countries with largest share of the global extreme poor, accordingly with WHO classifications, these countries are inhabited by people living on less than US$1.25 per day. Therefore, poverty levels show India 30%, Latin America 28%, China 8% and dramatically 20% represents African countries (Nigeria, Democratic Republic of the Congo, Ethiopia, United Republic of Tanzania, among others). Child mortality was 5.9 million children under age five which died in 2015, nearly 16,000 per day, mainly caused by infectious diseases whose distribution can be observed in Figure 1C. Main causes of child death are due to measles, malaria, diarrhea, HIV/AIDS, meningitis/encephalitis, tetanus and sepsis and other neonatal infections besides prematurity among other causes [1–3].
The sustainable development goals (SDGs) also contain ambitious targets for child mortality, with SDG 3.2 seeking to end preventable deaths of newborns and children under five. Those have included local aims for reducing the under-five mortality rates (U5MR) around to 25 deaths per 1000 live births as well as the neonatal mortality rate (NMR) to lower than 12 per 1000 live births, in comparison with a world’s U5MR rate of 43 per 1000 live births in the last year, representing 5.9 million deaths of children under 5 years and a NMR rate of 19 per 1000 live births, representing 2.7 million deaths in the first month of life. Main causes of newborn mortality during the last year were prematurity, birth-related complications and neonatal sepsis, while those post-neonatal causes of death were associated to pneumonia, diarrhea, injuries and malaria. Specifically, the so-called Target 4.2 in the document, which encourage for assuring that most children have access to good quality development, heath assistance and care, and basic education join to reducing child mortality while improving a better living quality for childhood in most poor countries .
On the other hand, Figure 1D displays climate changes and greenhouse effect on earth for a period between 1950 projected to the wear 2100. As described, global average surface temperature change is estimated under two scenarios for turning around global greenhouse gas emissions .
The global climate warming is a reality. The average data for the Earth’s surface temperature showed a 0.85°C increasing (0.65–1.06) for the 1880–2012 period. Data show that the Earth’s north hemisphere had the warmest period from 1983 to 2013, being the highest regarding the last 1400 years. Without any doubt, most causes for this fact can be associated to human activities. Mathematical and predictive algorithms for global warming-cooling allow establishing precise predictions on climate changes over long-time periods, these have included factors such as volcanic activity and gas emissions to the atmosphere. The Intergovernmental Panel on Climate Change’s (IPCC) for temperature changing prediction have considered a number of factors and possibilities for future greenhouse gas emissions, which have been termed as representative concentration pathways (RCP). This ranges from RPC 2.6 which considers that global greenhouse gas emissions will reach a highest value between 2010 and 2020, then it significantly decrease after 2020, to RCP 8.5, in which greenhouse gas emissions will continue to increase during the present century. Middle-range positions consider that RCP 4.5 and 6.0 would reach the highest emission values in 2040 and 2080 in consequence .
World’s predictive temperature changes for 2015–2016 period regarding those recorded between 1986 and 2005 are estimated to vary between 0.3 and 0.7°C. Similarly, increasing temperature ranges for the 2081–2100 period regarding the recorded changes between 1986 and 2005 has been estimated to be 0.3–1.7°C (RCP 2.6) to 2.6–4.8°C (RCP 8.5) (Figure 1D). In consequence, the Arctic region’s warming rate will increase faster than the world’s mean, and that for the land’s rate will be higher than the mean for the ocean. Assessed RPCs led to estimate that sea level will growth from 0.26 to 0.82 m by the final of the current age. Earth’s surface warming and climate variations will have a deep impact on human living, health and welfare, since obtaining drinking water and the possibility of cultivating the necessary quantities of agricultural products and all resources required for the future world’s larger population will be compromised.
3. Malaria: a devastating disease
Malaria is a global disease responsibly of high levels of morbidity and mortality especially in developing countries whose inhabitant populations suffer the consequences of the disease besides the economic impact on these populations. At the beginning of the new millennium, a global strategy for controlling malaria by establishing a global founding for fighting three high impact diseases, i.e., AIDS, tuberculosis and malaria have been proposed by the World Health Organization (WHO) . The 2015 world malaria report from WHO account data from 79 countries affected by this disease reflecting a slight improvement in controlling the disease impact but the problem still remains for a solution. In 2013, diagnosis tests were expanded to most malaria affecting countries and huge steps towards vector control were also conducted. In 2013, the use of insecticides impregnated mosquito nets were promoted and so the amount of populations protected against malaria were increased, thus mortality due to malaria was reduced to 47% between the years 2000 and 2013. However, endemic areas are still far away from reaching a total coverage for malaria control and available founding is each time decreased for managing this important problem. An estimated 278 million people in Africa live in households without a single insecticide mosquito net and 15 million pregnant women have no access to a preventive treatment for malaria. In addition, other diseases affecting these populations alter the development of related campaigns is the case of Ebola whose recent outbreak have conducted to a decreasing in health assistance in those affected zones.
In the last five years, it is estimated that 584,000 deaths due to malaria have occurred (367,000–755,000) of which 78% were children under 5 years of age and 90% came from Africa; today, there are an estimated 3.2 billion people at risk of contracting the disease since are living in areas influenced by the disease, of which 1.2 billion are at high risk (more than 1 into 1000 possibility of acquiring malaria in the year); in the Region of the Americas, it is presumed that the risk is 120 million people in 21 countries in the region .
Eradication efforts by public health preventive measures are not sufficiently effective for many reasons, among which are the socioeconomic, demographic and technical policies, emerging resistance to insecticides by the vector and to antimalarial drugs by the parasite . In 2010, vector resistance had been reported in 49 countries around the world of which 39 reported resistance above two or more pyrethroid insecticides. In 2013, this report increased to 82 countries reporting insecticide resistance , therefore, to develop an effective vaccine against the disease becomes an urgent need.
By 1967, major efforts were made to find an effective vaccine against human malaria, in one of the most important related studies of the time, 59% protection was achieved after an intravenous challenge of a malaria murine model after being vaccinated with 75,000 live attenuated irradiated sporozoites .
Currently among vaccine candidates that are in more advanced clinical trials are the RTS,S and
As can be observed in Figure 2, global malaria spreading accounts for more than 80 countries that are affected by malaria infection (purple background in the map). Besides, insecticide susceptibility status for malaria vectors (
Plasmodium spp. life cycle
A deep knowledge and understanding of the
There are five species of
In the human host, sporozoites are inoculated by the bite of female
The invasion of erythrocytes occurs after several steps with multiple interactions between receptor membrane proteins of host cells and parasite protein ligands expressed in its surface as well as in rhoptries and micronemes . The parasite grows and divides in about 72–48 h according to the specie to the schizont stage which contains more than 30 merozoite particles, which are released with the subsequent invasion and replication in healthy erythrocytes . Acquired immune response induced by malaria parasites is complex and varies depending on the level of endemicity, epidemiology, genetic, age of the host, parasitic stage and parasite species. Repeated infections and continued exposure are required to achieve clinical immunity with symptom reduction and reduced number of parasites in an infected individual or inhibition of parasite replication .
5. The murine model in the search for vaccine candidates against malaria
The mouse model has been widely used in the study on malaria, and it has been regarded as a practical model for experimental studies since its genetical features regarding human beings such as homology and similarity at the protein structure level, physiology and life cycle besides of owning a malaria transmission vector (
Due to this, there are several
The rodent malaria infection by
In studies at the level of liver infection cycle were found that about 654 (92%) of proteins in
Bearing in mind, the possibilities offered by murine infection models, we have conducted an important amount of experiments in order to test a variety of chemically modified antigens as potential vaccine components.
In spite of impressive economic and political efforts conducted by WHO and other non-government organizations for malaria eradication and control, based on insecticide treatment of bed-nets (mainly DTT), use of new formulations of artemisinin and other antimalarials for treatment of infected patients and teaching about an appropriate water and environment care to inhabitants of malaria high-transmission areas, among other strategies, malaria still remains as one of the most important health problems for developing countries. Contrary to those expectations, most of these strategies have failed for malaria control, mainly due to novel and powerful biological evolution of antimalarials-resistance mechanisms developed by
Up-to-date, about 236 including chemoprophylaxis and malaria vaccines clinical trials are being conducted worldwide, most of them have been completed showing a limited success (as shown in Figure 2). Most conducted studies have been focused on vaccine candidates aimed to block three different potential targets, being the transmission-blocking approach the first (gametocyte-derived proteins such as Pf25 and Pf125); secondly, those candidates directed against
As recently mentioned by Birkett in 2015, the European Medicines Agency announced a positive opinion for the malaria vaccine candidate most advanced in development, RTS,S/AS01, which provides modest protection against clinical malaria in all conducted trials, but in spite of its poor efficacy later in 2016, this product was recommended by WHO for large-scale trials in moderate to high malaria transmission areas . As observed in Figure 2, 113 trials of pharmaceutical products among antimalarials and vaccine formulations are being conducted in Africa in high-transmission malaria regions by immunizing mainly with modified or attenuated sporozoite NF54 strain malarial parasites or other products such as the so-named biological
Due to the moderate success conducted in the last three decades of researching for finding highly potent vaccines for preventing malaria, the field is open for new ideas regarding the discovery of strategies for developing structurally modulated molecular probes which address the
6. Current status of
P. vivax vaccine progress
Morbidity to malaria outside of the sub-Saharan Africa still remains meaningful causing more than 50% of malaria cases, especially in the Americas and Pacific-Asia where poverty and public health systems are associate to multiple problems. The complex
For multiple reasons, the epidemiologic spreading of malaria due to
On the other hand, the most focused
Therefore, developing potent
7. The meaning of being non-visible to α/β-TCR of T lymphocytes
It is well known the fact that the T-cell receptor sees antigen on the surface of cells associated with an MHC class I or II molecule. Therefore, activating humoral and cell-mediated immune responses requires factors such as cytokines and costimulatory molecules expressed by Th cells. A fine and specific regulation of Th has to be highly regulated in order to avoid any self-reactivity would conduct to auto-immune disorders. In order to ensure the Th-cells activation and regulation, these have to recognize a given antigen that is being presented in the MHC class-II context which is located on an antigen presenting cell (APC) surface. As it is known, these professional presenting cells among macrophages, dendritic cells and B lymphocytes harbor two relevant features: (1) surface expression of class-II (MHC-II) molecules, and (2) recruitment of costimulatory molecules as signals for activation of Th-cells.
Antigen-presenting cells first internalize antigen, and then display a part of that antigen on their membrane bound to a MHC-II molecule. The TH cell recognizes and interacts with the antigen–MHC-II molecule complex on the membrane of the antigen-presenting cell. Immune system is prepared for antigen presentation by stabilizing MHC-II molecules in the endoplasmic reticulum bound to an endogen invariant Ii chain which is later cleaved to a small peptide called class II-associated invariant chain peptide (CLIP) which remains bound to the MHC-II molecule to be then replaced by a given antigen-peptide assisted by a chaperone molecule named HLA-DM in endosomal compartments. Therefore, the antigen-MHC-II bimolecular complex will travel to the APC membrane surface to be presented to T-cell receptors (TCR) of T-lymphocytes to establish and stabilize in consequence specific ternary complexes able to trigger CD4+TH cell proliferation and so an immune response.
As mentioned one of the main functions of CLIP is to prevent the binding of self-peptide fragments prior to the MHC II localization within the endosome-lysosome, a consensus primary structure of CLIP is 87PVSKMRMATPLLMQA101, which is able to a proper interaction with a HLA-II molecule by anchoring-specific residues to the so-named pockets 1, 3, 4 and 9 of the MHC-II molecule in such a way that its entire structure will remain buried into the HLA-II molecule. The CLIP-HLA-II (CLIP: HLA-DR3) molecular complex is shown in Figure 4. As observed, the endogenous peptide is hidden into the presenting HLA-II molecule, and the possibility of being recognized by any TCR is completely abolished, and so an auto-reactive immune response will not take place, thus if a given pathogen can develop immune response evasion mechanisms based on its ligands structure features, it would be desirable to its convenience to resemble the most relevant structure characteristics of CLIP to avoid be recognized by TCRs .
8. How to become recognized by a TCR-T lymphocyte
Influenza hemagglutinin (HA) or hemagglutinin is a glycoprotein found on the surface of influenza viruses. Its role is to bind the influenza viruses to their target cells through sialic acid, specifically to red blood cells and upper respiratory tract cells [33, 34]. Once the pH has been decreased, a second role of HA is to join the viral cover to formed endosomes. HA is an integral membrane glycoprotein expressed as homo-trimers which seem a barrel-like structure having around 13.5 nm in length. HA is confirmed by three monomers built into a alpha-helical core displaying spherical tips containing those sialic acid-binding motifs. HA is synthesized as monomeric units as precursor forms which are glycosylated and processed on protein maturation, to produce two shorter proteins called HA1 and HA2. The HA monomers are long helical chains attached to the cell membrane by HA2 and capped by HA1. Thus, HA has been responsible for stimulation of neutralizing antibodies which are proven to avoid influenza virus infection to its target cells, thus constituting an important molecular tool for infection control using mechanisms associated to ternary complex stabilization of HA-HLA-II α/β-TCR (CD4+) with specific HLA-DR4 alleles such as DRA*0101 and DRB1*0401 .
Figure 5 recreates the 3D structure of the HA-hemagglutinin-HLAII-TCR ternary complex. As observed, the HA306–318 peptide backbone whose amino acid sequence is PKYVKQNTLKLAT anchor-specific residues into the HLA-II 1, 3, 4 and 9 pockets and clearly expose some residues in positions 5, 8 and 7 to be recognized by α/β-TCR chains and so stabilizing the molecular complex. However, the HA peptide binds promiscuously and can be presented by most of the frequently occurring DR alleles. Therefore, CD4+ T-lymphocyte proliferation would lead a subsequent neutralizing antibody production able to block the influenza virus infection, being this molecular interaction an effective mechanism effectively used for the immune response. As this a number of similar immuno-reactive complexes have been described [35–40].
9. New modified vaccine components containing non-natural elements considering chiral and topochemical constraints
Current prototype vaccines against malaria are failed to the goal of protecting any individual living in high risk malaria transmission areas even the most promising such as RTS,S which have less than 30% effectivity. As discussed above, the complex life cycle of
The aim of our research is to produce back-bone modified immunogenic antigens which included non-natural elements such as chiral and peptide-bond substitutions directed to modulate the antigen 3D structure and stimulation of neutralizing antibodies. Our approach is based on low-polymorphic sequences of
One of our first approaches for antigen peptide backbone modification consisted in introducing topochemical elements into the selected antigen primary structure, which consisted in two key features, first the amino acids chirality and second the peptide backbone space orientation. As represented in Figure 6A, the N-terminus low polymorphic region of the
CD patterns for the D-enantiomer and the
In order to test the unique recognition of an antibody stimulated by a reduced amide peptido-mimetic in which the oxygen atom of the carbonyl group (–R–CO–NH–R′–) of a relevant peptide-bond was replaced with two hydrogen atoms to lead an analogue being the reduced form of it and herein named reduced amide peptido-mimetic ψ(–R–CH2–NH–R′–). Thus, two monoclonal immunoglobulins (mAb) were produced, one directed to
Therefore, a whole set of
In another set of experiments conducted based on the
As observed in Figure 7, a few number of animals vaccinated have controlled the
On the contrary, animals of the placebo-control and those vaccinated with the native sequence became faster infected and did not control the
In order to be consisting with our proposed molecular models, we decided to focus our attention in another relevant
As observed in Figure 8, the reactivity patterns of both antibodies by western blot analyses lead to identified native
In order to verify the antibody reactivity, an
Similarly, a lysate composed by membrane proteins from blood stages of
To obtain a complete landscape of this novel scenario, further scopes of the strategy of obtaining next generations of malaria vaccine candidates based on introducing non-natural elements into immunogens, trials performed in selected antigens of other
As reported before, a class-I restricted
The proposed hypothesis has been confirmed by challenging it in different molecular scenarios, all based on analysis of different antigens derived from different
Aimed to understand a possible structure-immunological activity relationship, a subsequent set of nuclear magnetic resonance NMR and molecular dynamic
As observed in Figure 10A, overlapped 3D conformations of the
Similarly, backbone of the low polymorphic
On the other hand, backbone structural analyses for the
An interesting observation become evident when backbone of two homologue proteins are overlapped regarding a class-I epitope region, as it was the case of the
Further experiments in this pursuit will explore hypothesis on
The family of the herein presented structural modified compounds constitute molecular tools to be considered for new generations of functional protective vaccines against malaria, as such, future vaccine candidates could be based on this knowledge and outstanding findings.
As the author of this work, I am indebted to Prof. Manuel Elkin Patarroyo for his invaluable contribution to my personal view on the malaria vaccine field. Special thanks to the Colombian Science Technology and Innovation Department (Colciencias) (Grant No. 212456934488).
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