ookluc-plasmodium-berghei-line-for-identifying-transmission-blocking

<em>Ookluc</em>: A <em>Plasmodium berghei</em> Line for Identifying Transmission-blocking Compounds

Malaria remains a world health challenge; in 2023, 263 million cases and 597,000 deaths were reported around the globe. The parasite's life cycle is ...

University of São Paulo

 is the most common species of human malaria parasite found outside Africa, with high endemicity in Asia, Central and South America, and Oceania. Although  causes the majority of deaths,  can lead to severe malaria and result in significant morbidity and mortality. The development of a protective vaccine will be a major step toward malaria elimination. Recently, a formulation containing the three allelic variants of the  circumsporozoite protein (PvCSP-All epitopes) showed partial protection in mice after a challenge with the hybrid  (Pb) sporozoite, in which the PbCSP central repeats were replaced by the VK210 PvCSP repeats (Pb/Pv sporozoite). In the present study, the chimeric PvCSP allelic variants (VK210, VK247, and -like) were fused with the mumps virus nucleocapsid protein in the absence (NLP-CSP) or presence of the conserved C-terminal (CT) domain of PvCSP (NLP-CSP). To elicit stronger humoral and cellular responses,  yeast was used to assemble them as nucleocapsid-like particles (NLPs). Mice were immunized with each recombinant protein adjuvanted with Poly (I:C) and presented a high frequency of antigen-specific antibody-secreting cells (ASCs) on days 5 and 30, respectively, in the spleen and bone marrow. Moreover, high IgG titers against all PvCSP variants were detected in the sera. Later, these immunized mice with NLP-CSP were challenged with Pb/Pv sporozoites. Sterile protection was observed in 30% of the challenged mice. Therefore, this vaccine formulation use has the potential to be a good candidate for the development of a universal vaccine against  malaria.

Protective Malaria Vaccine in Mice Based on the  Circumsporozoite Protein Fused with the Mumps Nucleocapsid Protein.

Infections with  are predominant in the Americas, representing 75% of malaria cases. Previously perceived as benign, malaria vivax is, in fact, a highly debilitating and economically important disease. Considering the high complexity of the malaria parasite life cycle, it has been hypothesized that an effective vaccine formulation against  should contain multiple antigens expressed in different parasite stages. Based on that, we analyzed a recombinant  vaccine formulation mixing the apical membrane antigen 1 ectodomain (PvAMA-1) and a full-length circumsporozoite protein (PvCSP-All) previously studied by our group, which elicits a potent antibody response in mice. Genetically distinct strains of mice (C57BL/6 and BALB/c) were immunized with the proteins, alone or in combination, in the presence of poly(I:C) adjuvant, a TLR3 agonist. In C57BL/6, high-antibody titers were induced against PvAMA-1 and the three PvCSP variants (VK210, VK247, and -like). Meanwhile, mixing PvAMA-1 with PvCSP-All had no impact on total IgG antibody titers, which were long-lasting. Moreover, antibodies from immunized mice recognized VK210 sporozoites and blood-stage parasites by immunofluorescence assay. However, in the BALB/c model, the antibody response against PvCSP-All was relatively low. PvAMA-1-specific CD3CD4 and CD3CD8 T-cell responses were observed in C57BL/6 mice, and the cellular response was impaired by PvCSP-All combination. More relevant, the multistage vaccine formulation provided partial protection in mice challenged with a transgenic  sporozoite expressing the homologous PvCSP protein.

A Multistage Formulation Based on Full-Length CSP and AMA-1 Ectodomain of  Induces High Antibody Titers and T-cells and Partially Protects Mice Challenged with a Transgenic  Parasite.

Malaria is a human parasitic disease distributed in many tropical countries and caused by various  species.  has the largest geographical distribution of the  species and is predominant in the Americas, including Brazil. Only a small number of  vaccine formulations have successfully reached clinical trials relative to their  counterparts. One of the candidate antigens for a blood-stage  vaccine is apical membrane antigen 1 (PvAMA-1). Due to the worldwide distribution of  parasites, a high degree of variability has been detected in this antigen sequence, representing a considerable challenge to the development of a universal vaccine against malaria. In this study, we evaluated how PvAMA-1 polymorphisms influence vaccine-derived immune responses in  malaria. To this end, we expressed 9 recombinant protein representatives of different PvAMA-1 allelic variants in the yeast : Belem, Chesson I, Sal-1, Indonesia XIX, SK0814, TC103, PNG_05_ESP, PNG_62_MU, and PNG_68_MAS. After protein expression and purification, we evaluated the breadth of the immune responses derived from malaria-exposed individuals from the Amazon region. From 611 serum samples of malaria-exposed individuals, 53.68% of them reacted against the PvAMA-1 Belem through ELISA. Positive samples were further tested against recombinant proteins representing the other PvAMA-1 allelic variants. Whereas Sal-1, Chesson I and SK0814 variants were highly recognized by tested serum samples, Indonesia XIX, TC103, PNG_05_ESP, PNG_62_MU, and PNG_68_MAS were only slightly recognized. Moreover, polyclonal sera derived from C57BL/6 mice immunized with the PvAMA-1 Belem protein predominantly recognized Belem, Sal-1, Chesson I, SK0814, and Indonesia XIX through ELISA. Last, ELISA-based competition assays demonstrated that a previous interaction between anti-Belem polyclonal serum and Sal-1, Chesson I, SK0814, or Indonesia XIX proteins could further inhibit antibody binding to the Belem variant. Our human and mouse data suggest the presence of common epitopes or cross-reactivity between Belem, Sal-1, Chesson I, and SK0814 variants. Although the PvAMA-1 Belem variant induces strain-transcendent antibodies, PvAMA-1 variants from Thailand and Papua New Guinea may need to be included in a universal vaccine formulation to achieve protection against  malaria.

Antibodies Against the  Apical Membrane Antigen 1 From the Belem Strain Share Common Epitopes Among Other Worldwide Variants.

Malaria is a serious public health problem that affects mostly the poorest countries in the world, killing more than 400,000 people per year, mainly children under 5 years old. Among the control and prevention strategies, the differential diagnosis of the -infecting species is an important factor for selecting a treatment and, consequently, for preventing the spread of the disease. One of the main difficulties for the detection of a specific  sp is that most of the existing methods for malaria diagnosis focus on detecting . Thus, in many cases, the diagnostic methods neglect the other non-falciparum species and underestimate their prevalence and severity. Traditional methods for diagnosing malaria may present low specificity or sensitivity to non-falciparum spp. Therefore, there is high demand for new alternative methods able to differentiate  species in a faster, cheaper and easier manner to execute. This review details the classical procedures and new perspectives of diagnostic methods for malaria non-falciparum differential detection and the possibilities of their application in different circumstances.

Diagnostic Methods for Non-Falciparum Malaria.

Malaria is a highly prevalent parasitic disease in regions with tropical and subtropical climates worldwide. Among the species of Plasmodium causing human malaria, P. vivax is the second most prevalent and the most geographically widespread species. A major target of a pre-erythrocytic vaccine is the P. vivax circumsporozoite protein (PvCSP). In previous studies, we fused two recombinant proteins representing three allelic variants of PvCSP (VK210, VK247 and P. vivax-like) to the mumps virus nucleocapsid protein to enhance immune responses against PvCSP. The objective of the present study was to evaluate the protective efficacy of these recombinants in mice challenged with transgenic P. berghei parasites expressing PvCSP allelic variants. Formulations containing Poly (I:C) or Montanide ISA720 as adjuvants elicited high and long-lasting IgG antibody titers specific to each PvCSP allelic variant. Immunized mice were challenged with two existing chimeric P. berghei parasite lines expressing PvCSP-VK210 and PvCSP-VK247. We also developed a novel chimeric line expressing the third allelic variant, PvCSP-P. vivax-like, as a new murine immunization-challenge model. Our formulations conferred partial protection (significant delay in the time to reach 1% parasitemia) against challenge with the three chimeric parasites. Our results provide insights into the development of a vaccine targeting multiple strains of P. vivax.

A universal vaccine candidate against Plasmodium vivax malaria confers protective immunity against the three PvCSP alleles.

Adjuvants are essential for vaccine development, especially subunit-based vaccines such as those containing recombinant proteins. Increasing the knowledge of the immune response mechanisms generated by adjuvants should facilitate the formulation of vaccines in the future. The present work describes the immune phenotypes induced by Poly (I:C) and Montanide ISA 720 in the context of mice immunization with a recombinant protein based on the circumsporozoite protein (PvCSP) sequence. Mice immunized with the recombinant protein plus Montanide ISA 720 showed an overall more robust humoral response, inducing antibodies with greater avidity to the antigen. A general trend for mixed Th1/Th2 inflammatory cytokine profile was increased in Montanide-adjuvanted mice, while a balanced profile was observed in Poly (I:C)-adjuvanted mice. Montanide ISA 720 induced a gene signature in B lymphocytes characteristic of heme biosynthesis, suggesting increased differentiation to Plasma Cells. On the other hand, Poly (I:C) provoked more perturbations in T cell transcriptome. These results extend the understanding of the modulation of specific immune responses induced by different classes of adjuvants, and could support the optimization of subunit-based vaccines.

Immune System Modulation by the Adjuvants Poly (I:C) and Montanide ISA 720.

In 2019, the world witnessed the onset of an unprecedented pandemic. By February 2022, the infection by SARS-CoV-2 has already been responsible for the death of more than 5 million people worldwide. Recently, we and other groups discovered that SARS-CoV-2 infection induces ER stress and activation of the unfolded protein response (UPR) pathway. Degradation of misfolded/unfolded proteins is an essential element of proteostasis and occurs mainly in lysosomes or proteasomes. The N-terminal arginylation of proteins is characterized as an inducer of ubiquitination and proteasomal degradation by the N-degron pathway.

Protein Arginylation Is Regulated during SARS-CoV-2 Infection.

Malaria caused byPlasmodium vivaxis a pressing public health problem in tropical and subtropical areas.However, little progress has been made toward developing a P. vivaxvaccine, with only three candidates being tested in clinical studies. We previously reported that one chimeric recombinant protein (PvCSP-All epitopes) containing the conserved C-terminus of the P. vivax Circumsporozoite Protein (PvCSP), the three variant repeat domains, and aToll-like receptor-3 agonist,Poly(I:C), as an adjuvant (polyinosinic-polycytidylic acid, a dsRNA analog mimicking viral RNA), elicits strong antibody-mediated immune responses in mice to each of the three allelic forms of PvCSP. In the present study, a pre-clinical safety evaluation was performed to identify potential local and systemic toxic effects of the PvCSP-All epitopes combined with the Poly-ICLC (Poly I:C plus poly-L-lysine, Hiltonol®) or Poly-ICLC when subcutaneously injected into C57BL/6 mice and New Zealand White Rabbits followed by a 21-day recovery period. Overall, all observations were considered non-adverse and were consistent with the expected inflammatory response and immune stimulation following vaccine administration. High levels of vaccine-induced specific antibodies were detected both in mice and rabbits. Furthermore, mice that received the vaccine formulation were protected after the challenge with Plasmodium berghei sporozoites expressing CSP repeats from P. vivax sporozoites (Pb/Pv-VK210). In conclusion, in these non-clinical models, repeated dose administrations of the PvCSP-All epitopes vaccine adjuvanted with a Poly-ICLC were immunogenic, safe, and well tolerated.

Non-clinical toxicity and immunogenicity evaluation of a Plasmodium vivax malaria vaccine using Poly-ICLC (Hiltonol®) as adjuvant.

Malaria remains a global health challenge, necessitating the development of effective vaccines. The RTS,S vaccination prevents  (Pf) malaria but is ineffective against  (Pv) disease. Herein, we evaluated the murine immunogenicity of a recombinant PvCSP incorporating prevalent polymorphisms, adjuvanted with Alhydrogel or Poly I:C. Both formulations induced prolonged IgG responses, with IgG1 dominance by the Alhydrogel group and high titers of all IgG isotypes by the Poly I:C counterpart. Poly I:C-adjuvanted vaccination increased splenic plasma cells, terminally-differentiated memory cells (MBCs), and precursors relative to the Alhydrogel-combined immunization. Splenic B-cells from Poly I:C-vaccinated mice revealed an antibody-secreting cell- and MBC-differentiating gene expression profile. Biological processes such as antibody folding and secretion were highlighted by the Poly I:C-adjuvanted vaccination. These findings underscore the potential of Poly I:C to strengthen immune responses against Pv malaria.

Poly I:C elicits broader and stronger humoral and cellular responses to a  circumsporozoite protein malaria vaccine than Alhydrogel in mice.

Malaria, a life-threatening disease caused by Plasmodium parasites, continues to pose a significant global health threat, with nearly 250 million infections and over 600 000 deaths reported annually by the WHO. Fighting malaria is particularly challenging partly due to the complex life cycle of the parasite. However, technological breakthroughs such as the development of the nucleoside-modified mRNA lipid nanoparticle (mRNA-LNP) vaccine platform, along with the discovery of novel conserved Plasmodium antigens such as the E140 protein, present new opportunities in malaria prevention. Importantly, production of recombinant proteins for malaria vaccine evaluation by serological assays often represents an additional hurdle because many Plasmodium proteins are complex and often contain transmembrane domains that make production and purification particularly difficult. This research protocol provides a step-by-step guide for the production and purification of P. berghei and P. vivax E140 protein fragments that can be used to test humoral immune responses against this novel malaria vaccine target. We demonstrate that the purified proteins can be successfully used in enzyme-linked immunosorbent assay (ELISA) to evaluate antigen-specific binding antibody responses in sera obtained from E140 mRNA-LNP-vaccinated mice. Therefore, these proteins can contribute to the development and evaluation of E140-based malaria vaccines.

Expression and purification of E140 protein antigen fragments of Plasmodium vivax and Plasmodium berghei for serological assays.

COVID-19 is a human respiratory syndrome caused by the infection of the SARS-CoV-2 virus that has a high rate of infection and mortality. Viruses modulate the host machinery by altering cellular mechanisms that favor their replication. One of the mechanisms that viruses exploit is the protein folding and processing of post-translational modifications that occur in the endoplasmic reticulum (ER). When ER function is impaired, there is an accumulation of misfolded proteins leading to endoplasmic reticulum stress (ER stress). To maintain homeostasis, cells trigger an adaptive signaling mechanism called the Unfolded Protein Response (UPR) which helps cells deal with stress, but under severe conditions, can activate the apoptotic cell death mechanism. This study elucidated an activation of a diversity of molecular mechanisms by Brazilian variants of SARS-CoV-2 by a time-resolved and large-scale characterization of SARS-CoV-2-infected cells proteomics and immunoblotting. Furthermore, it was shown that pharmacological UPR modulation could reduce viral release by counteracting the different viral activations of its cellular response. Analysis of human clinical specimens and disease outcomes focusing on ER stress reinforces the importance of UPR modulation as a host regulatory mechanism during viral infection and could point to novel therapeutic targets. SIGNIFICANCE: Since the emergence of SARS-CoV-2 and the consequent COVID-19 pandemic, the rapid emergence of variants of this new coronavirus has been a cause for concern since many of them have significantly higher rates of transmissibility and virulence, being called Variants of Concern (VOC). In this work, we studied the VOCs Gamma (P.1) and Zeta (P.2), also known as Brazilian variants. Constant evidence has reported that there are particularities related to each variant of SARS-CoV-2, with different rates of transmissibility, replication and modulation of host biological processes being observed, in addition to the mutations present in the variants. For this reason, this work focused on infections caused by the Brazilian variants of SARS-CoV-2 in different cell lines, in which we were able to observe that the infections caused by the variants induced endoplasmic reticulum stress in the infected cells and activated the UPR pathways, presenting specific modulations of each variant in this pathway. Furthermore, transcriptome analysis of patients revealed a correlation between ER-related genes and COVID-19 progression. Finally, we observed that the use of UPR modulators in host cells decreased viral release of all variants without affecting cell viability. The data presented in this work complement the observations of other studies that aim to understand the pathogenicity of SARS-CoV-2 VOCs and possible new therapeutic strategies, mainly targeting biological processes related to the endoplasmic reticulum.

Article	Year
Protective Malaria Vaccine in Mice Based on the Circumsporozoite Protein Fused with the Mumps Nucleocapsid Protein. Vaccines\| PubMed ID: 32325874	2020
A Multistage Formulation Based on Full-Length CSP and AMA-1 Ectodomain of Induces High Antibody Titers and T-cells and Partially Protects Mice Challenged with a Transgenic Parasite. Microorganisms\| PubMed ID: 32560380	2020
Antibodies Against the Apical Membrane Antigen 1 From the Belem Strain Share Common Epitopes Among Other Worldwide Variants. Frontiers in cellular and infection microbiology\| PubMed ID: 33796476	2021
Diagnostic Methods for Non-Falciparum Malaria. Frontiers in cellular and infection microbiology\| PubMed ID: 34222049	2021
A universal vaccine candidate against Plasmodium vivax malaria confers protective immunity against the three PvCSP alleles. Scientific reports\| PubMed ID: 34504134	2021
Immune System Modulation by the Adjuvants Poly (I:C) and Montanide ISA 720. Frontiers in immunology\| PubMed ID: 35844531	2022
Protein Arginylation Is Regulated during SARS-CoV-2 Infection. Viruses\| PubMed ID: 36851505	2023
Non-clinical toxicity and immunogenicity evaluation of a Plasmodium vivax malaria vaccine using Poly-ICLC (Hiltonol®) as adjuvant. Vaccine\| PubMed ID: 38448321	2024
Poly I:C elicits broader and stronger humoral and cellular responses to a circumsporozoite protein malaria vaccine than Alhydrogel in mice. Frontiers in immunology\| PubMed ID: 38650939	2024
Expression and purification of E140 protein antigen fragments of Plasmodium vivax and Plasmodium berghei for serological assays. FEBS open bio\| PubMed ID: 39815669	2025
P.1 and P.2 SARS-CoV-2 Brazilian variants activate the unfolded protein response with a time and pathway specificity. Journal of proteomics\| PubMed ID: 39909104	2025

Rodolfo F. Marques

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