Plasmodium vivax pre-erythrocytic vaccines.

An estimated 229 million cases of malaria occurred worldwide in 2019. Both, Plasmodium falciparum and P. vivax are responsible for most of the malaria disease burden in the world. Despite difficulties in obtaining an accurate number, the global estimates of cases in 2019 are approximately 229 million of which 2.8% are due to P. vivax, and the total number of malaria deaths are approximately 409 million. Regional elimination or global eradication of malaria will be a difficult task, particularly for P. vivax due to the particular biological features related to the hypnozoite, leading to relapse. Countries that have shown successful episodes of a decrease in P. falciparum malaria, are left with remaining P. vivax malaria cases. This is caused by the mechanism that the parasite has evolved to remain dormant in the liver forming hypnozoites. Furthermore, while clinical trials of vaccines against P. falciparum are making fast progress, a very different picture is seen with P. vivax, where only few candidates are currently active in clinical trials. We discuss the challenge that represent the hypnozoite for P. vivax vaccine development, the potential of Controlled Human Malaria Challenges (CHMI) and the leading vaccine candidates assessed in clinical trials.

Building momentum for malaria vaccine research and development: key considerations.

To maintain momentum towards improved malaria control and elimination, a vaccine would be a key addition to the intervention toolkit. Two approaches are recommended: (1) promote the development and short to medium term deployment of first generation vaccine candidates and (2) support innovation and discovery to identify and develop highly effective, long-lasting and affordable next generation malaria vaccines.

Commentary in reply to a publication on Plasmodium falciparum pre-erythrocytic stage vaccine development.

We have read the publication of Molina-Franky and colleagues on Plasmodium falciparum pre-erythrocytic stage vaccine development (Malaria Journal, 2020;19:56). The commentary revises some of their statements on the RTS,S/AS01 vaccine that are considered either imprecise or incorrect.

Disulfide bond mapping of Pfs25, a recombinant malaria transmission blocking vaccine candidate.

A liquid chromatography tandem-mass spectrometry method was developed to map the eleven disulfide bonds in Pfs25, a malaria transmission-blocking vaccine candidate. The compact and complex nature of Pfs25 has led to difficulties in prior peptide mapping efforts. Here, we report confirmation of proper disulfide pairing of a recombinant Pfs25, by optimizing denaturation and digestion with trypsin/Lys-C. The digested peptides were separated by reversed phase HPLC to obtain the peptide map and elucidate the disulfide linkages. MSE fragmentation confirmed the digested peptides and disulfide bonds. The eleven disulfide bonds and locations matched the predicted Pvs25 crystal structure, a Pfs25 homologue.

Plasmodium knowlesi: a relevant, versatile experimental malaria model.

The primate malaria Plasmodium knowlesi has a long-standing history as an experimental malaria model. Studies using this model parasite in combination with its various natural and experimental non-human primate hosts have led to important advances in vaccine development and in our understanding of malaria invasion, immunology and parasite-host interactions. The adaptation to long-term in vitro continuous blood stage culture in rhesus monkey, Macaca fascicularis and human red blood cells, as well as the development of various transfection methodologies has resulted in a highly versatile experimental malaria model, further increasing the potential of what was already a very powerful model. The growing evidence that P. knowlesi is an important human zoonosis in South-East Asia has added relevance to former and future studies of this parasite species.

Western blot assay for quantitative and qualitative antigen detection in vaccine development.

Immunological methods for quantitative measurement, antigenic characterization, and monitoring the stability of active immunogenic component(s) are a critical need in the vaccine development process. This unit describes an enhanced chemiluminescence-based western blot for quantitative detection of Plasmodium falciparum circumsporozoite protein (PfCSP), a major malaria candidate vaccine antigen. The most salient features of this assay are its high sensitivity and reproducibility; it can reliably detect ∼5 to 10 pg PfCSP expressed on native parasites or recombinantly expressed in Escherichia coli. Although described for a specific vaccine antigen, this assay should be applicable for any antigen-antibody combination for which relevant detection reagents are available. Detailed stepwise experimental procedures and methods for data acquisition and analysis are described.

Use of o-phthalaldehyde assay to determine protein contents of Alhydrogel-based vaccines.

Aluminum based adjuvants (alum), including aluminum hydroxide (Alhydrogel) and aluminum phosphate are the most commonly used adjuvant in the US. In order to ensure quality of vaccines, regulatory authorities require evaluation of antigen content in final vaccine products. Currently, there are no generic methods available for the determination of protein content in alum-based vaccines. Aluminum hydroxide gels exist as particles in solution, which interfere with direct quantitation of protein content in formulations using assays such as Lowry, BCA or Bradford protein assay. The present study adapts a simple fluorescent assay to directly (without the need for antigen extraction) determine antigen content on Alhydrogel with accuracy and sensitivity using the o-phthalaldehyde (OPA) reagent. Malaria vaccine candidates AMA1-C1/Alhydrogel, AMA1-C2/Alhydrogel, MSP1(42)-3D7/Alhydrogel, MSP1(42)-C1/Alhydrogel or BSAM-2/Alhydrogel were used as model formulations. The results of the present study show that the OPA assay is highly accurate (87-100%), reproducible, and simple with a linear detection range of 25-400 microg/mL for Alhydrogel vaccines (except for MSP1(42)-C1, which has a linear detection range of 31.25-500 microg/mL). This assay has proven to be highly useful in our laboratory and been used in routine vaccine quality control processes.

Vacuna frente a la malaria: el gran reto para los países en vías de desarrollo / Malaria vaccine: the main challenge for developing countries

La malaria, enfermedad causada por parásitosprotozoarios del género Plasmodium, representa uno delos problemas más graves de salud pública en el mundo yes la enfermedad parasitaria más importante en humanos.Se estima que unos 3.000 millones de personas estánexpuestas a la enfermedad al vivir en zonas endémicas, ycada año ocurren entre 300-500 millones de episodiosclínicos de los cuales aproximadamente 1-3 millonesmueren, la mayoría niños menores de 5 años. A pesar deser una enfermedad endémica en varios continentes, es enÁfrica, y especialmente en los territorios comprendidosentre los 2 trópicos, donde se acumula hasta un 90% de las muertes, casi siempre en niños menores de 5 años. Existen diferentes y complementarias herramientas disponibles para controlar la enfermedad, entre las que destacan las siguientes: el diagnóstico e inicio rápido del tratamiento de los casos con un antimalárico eficaz, la disminución del contacto entre hombre y vector, fundamentalmente con redes mosquiteras impregnadas de insecticida, el tratamiento preventivo intermitente en niños y mujeres embarazadas, y el control del vector mediante fumigación intradomiciliaria o larvicidas. Sin embargo, la implementación de estos mecanismos de control sigue siendo incompleta en la mayoría de las zonas endémicas.Una vacuna eficaz y segura en niños y de bajo coste,sumado a las demás medidas de control ya existentes,sería un elemento clave en el control de la enfermedad. La vacuna que se encuentra en fases más avanzadas deinvestigación es la compuesta por un antígenopreeritrocítico CSP, llamado RTS,S. Esta vacuna demostróseguridad, immunogenicidad y eficacia en niños de un árearural de Mozambique. Los enormes progresos que se hanhecho en los últimos años, junto con unos primerosresultados muy esperanzadores, permiten un optimismorazonable acerca del desarrollo de una vacuna en un futuro próximo

Malaria, which is caused by protozoan parasites of thePlasmodium genus, is one of the most serious publichealth problems worldwide and is the most importantparasitic infection in humans. Approximately 3,000 million people are estimated to be exposed to the disease by living in endemic areas. Every year, there are between 300 and 500 million clinical episodes, causing approximately 1-3 million deaths, mainly in children aged less than 5 years. Although malaria is endemic in several continents, 90% of deaths occur in Africa, especially in the regions between the two tropics, and most of those who die areyoung children. Several tools, which are complementary,are available to control the disease, notable among whichare the following: diagnosis and prompt treatment with aneffective antimalarial agent, reduction of contact between humans and vector –mainly through the use of insecticideimpregnated mosquito nets–, intermittent preventive treatment in children and pregnant women, and vector control through fumigation in the home or the use of larvicides. However, the implementation of these control measures continues to be incomplete in most endemic areas. In addition to already existing control measures, a safe, effective and lowcost vaccine in children would be a key element in controlling the disease. The vaccine in the most advanced phases of research is that composed by a preerythrocytic antigen, circumsporozoite protein (CSP), called RTS,S. This vaccine has been shown to be safe, immunogenic and effective in children in a rural area of Mozambique. Given the huge advances that have takenplace in the last few years, together with highlyencouraging initial results, optimism about thedevelopment of a vaccine in the near future is reasonable

Improvement in yield and purity of a recombinant malaria vaccine candidate based on the receptor-binding domain of Plasmodium vivax Duffy binding protein by codon optimization.

A recombinant blood-stage vaccine for Plasmodium vivax malaria based on the functional receptor-binding domain of PvDBP (PvRII) has been developed. A synthetic gene coding for PvRII was expressed in Escherichia coli using codon optimization. Expression level of recombinant PvRII was 10% of the total cellular proteins. Truncated PvRII products, seen when the native PvRII gene was expressed, were absent in case of synthetic gene.

Avances en vacunas contra la malaria / Advances in vaccines against malaria

Los últimos avances en el desarrollo de una vacuna contra la malaria van encaminados a conseguir que ésta sea a la vez segura e inmunógena. Actualmente existen diversos tipos de vacunas en estudio, dirigidas hacia las distintas fases del ciclo vital del Plasmodium. De esta manera se han desarrollado vacunas que actúan en la fase asexual (pre y eritrocítica) y en la fase sexual (bloqueo de la transmisión) del parásito. Hemos revisado los últimos datos obtenidos sobre estas líneas de investigación y las dificultades que se presentan para la obtención de una vacuna eficaz

The last advances in the development against malaria vaccines are going to get a safe and immunogenic vaccines. At the moment there are several types of vaccines in investigation, they are directed at the different phases of the vital cycle of Plasmodium. So vaccines have been developed to actuating in the asexual phase (pre and erytrocytic vaccines) and in the sexual phase (transmission block vaccines) parasite. We have reviewed the last studies and the problems to achieve an efficient vaccine

Enhanced expression of a recombinant malaria candidate vaccine in Escherichia coli by codon optimization.

This study was conducted to compare the expression of three constructs of a multistage candidate vaccine (FALVAC-1) against Plasmodium falciparum in an Escherichia coli system: a synthetic gene with P. falciparum codons, a synthetic gene with optimized E. coli codons, and a synthetic gene with P. falciparum codons co-transformed with a RIG plasmid, which encodes three tRNAs (AG(A/G), ATA, GGA) that recognize rare E. coli codons. The expression of the protein increased at least threefold with codon optimization. The presence of the RIG plasmid in the co-transforming cells did not significantly increase the expression level of the gene with P. falciparum codons. The growth of cells transformed by the construct with P. falciparum codons was significantly slower than that of cells transformed by the construct with optimized E. coli codons after induction of protein expression with IPTG. The cells containing the non-codon optimized gene co-expressed with RIG plasmid had the slowest growth at all time points in culture. Thus, codon optimization significantly increases the yield of P. falciparum candidate vaccines in the E. coli expression system.