Monica Cabrera-Mora

Plasmodium coatneyi in Rhesus Macaques Replicates the Multisystemic Dysfunction of Severe Malaria in Humans

ABSTRACT Severe malaria, a leading cause of mortality among children and nonimmune adults, is a multisystemic disorder characterized by complex clinical syndromes that are mechanistically poorly understood. The interplay of various parasite and host factors is critical in the pathophysiology of severe malaria. However, knowledge regarding the pathophysiological mechanisms and pathways leading to the multisystemic disorders of severe malaria in humans is limited. Here, we systematically investigate infections with Plasmodium coatneyi, a simian malaria parasite that closely mimics the biological characteristics of P. falciparum, and develop baseline data and protocols for studying erythrocyte turnover and severe malaria in greater depth. We show that rhesus macaques (Macaca mulatta) experimentally infected with P. coatneyi develop anemia, coagulopathy, and renal and metabolic dysfunction. The clinical course of acute infections required suppressive antimalaria chemotherapy, fluid support, and whole-blood transfusion, mimicking the standard of care for the management of severe malaria cases in humans. Subsequent infections in the same animals progressed with a mild illness in comparison, suggesting that immunity played a role in reducing the severity of the disease. Our results demonstrate that P. coatneyi infection in rhesus macaques can serve as a highly relevant model to investigate the physiological pathways and molecular mechanisms of malaria pathogenesis in naïve and immune individuals. Together with high-throughput postgenomic technologies, such investigations hold promise for the identification of new clinical interventions and adjunctive therapies.

Genetic linkage of autologous T cell epitopes in a chimeric recombinant construct improves anti-parasite and anti-disease protective effect of a malaria vaccine candidate

We have reported the design of polyvalent synthetic and recombinant chimeras that include promiscuous T cell epitopes as a viable delivery system for pre-erythrocytic subunit malaria vaccines. To further assess the ability of several Plasmodium T cell epitopes to enhance vaccine potency, we designed a synthetic gene encoding four Plasmodium yoelii merozoite surface protein 1 (PyMSP1) CD4(+) promiscuous T cell epitopes fused in tandem to the homologous carboxyl terminal PyMSP1(19) fragment. This Recombinant Modular Chimera (PyRMC-MSP1(19)) was tested for immunogenicity and protective efficacy in comparative experiments with a recombinant protein expressing only the PyMSP1(19) fragment. Both proteins induced comparable antibody responses. However PyRMC-MSP1(19) elicited higher anti-parasite antibody titers and more robust protection against both hyper-parasitemia and malarial anemia. Most importantly, passive transfer of anti-PyRMC-MSP1(19), but not anti-PyMSP1(19) antibodies protected against heterologous challenge. These studies show that protective efficacy can be significantly improved by inclusion of an array of autologous promiscuous T cell epitopes in vaccine constructs.

A Hybrid Multistage Protein Vaccine Induces Protective Immunity against Murine Malaria

ABSTRACT We have previously reported the design and expression of chimeric recombinant proteins as an effective platform to deliver malaria vaccines. The erythrocytic and exoerythrocytic protein chimeras described included autologous T helper epitopes genetically linked to defined B cell epitopes. Proof-of-principle studies using vaccine constructs based on the Plasmodium yoelii circumsporozoite protein (CSP) and P. yoelii merozoite surface protein-1 (MSP-1) showed encouraging results when tested individually in this mouse malaria model. To evaluate the potential synergistic or additive effect of combining these chimeric antigens, we constructed a synthetic gene encoding a hybrid protein that combined both polypeptides in a single immunogen. The multistage vaccine was expressed in soluble form in Escherichia coli at high yield. Here we report that the multistage protein induced robust immune responses to individual components, with no evidence of vaccine interference. Passive immunization using purified IgG from rabbits immunized with the hybrid protein conferred more robust protection against the experimental challenge with P. yoelii sporozoites than passive immunization with purified IgG from rabbits immunized with the individual proteins. High antibody titers and high frequencies of CD4+- and CD8+-specific cytokine-secreting T cells were elicited by vaccination. T cells were multifunctional and able to simultaneously produce interleukin-2 (IL-2), gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α). The mechanism of vaccine-induced protection involved neutralizing antibodies and effector CD4+ T cells and resulted in the control of hyperparasitemia and protection against malarial anemia. These data support our strategy of using an array of autologous T helper epitopes to maximize the response to multistage malaria vaccines.

Recombinant peptide replicates immunogenicity of synthetic linear peptide chimera for use as pre-erythrocytic stage malaria vaccine

Synthetic linear peptide chimeras (LPCs(cys+)) show promise as delivery platforms for malaria subunit vaccines. Maximal immune response to LPCs(cys+) in rodent malaria models depends upon formation of cross-linkages to generate homopolymers, presenting challenges for vaccine production. To replicate the immunogenicity of LPCs(cys+) using a recombinant approach, we designed a recombinant LPC (rLPC) based on Plasmodium yoelii circumsporozoite protein-specific sequences of 208 amino acids consisting of four LPC subunits in series. BALB/c or CAF1/J mice were immunized with synthetic or recombinant LPCs. Antibody concentrations, cytokine production and protection against challenge were compared. Recombinant peptide replicated the robust, high avidity antibody responses obtained with the synthetic linear peptide chimera. After in vitro stimulation spleen cells from mice immunized with rLPC or synthetic LPC(cys+) produced gamma interferon and IL-4 suggesting the efficient priming of T cells. Immunization of mice with either recombinant or synthetic LPC(cys+) provided comparable protection against experimental challenge with P. yoelii sporozoites. Recombinant LPCs reproduced the immunogenicity of synthetic LPC(cys+) without requiring polymerization, improving prospects for use as malaria vaccines.

Evaluation of naturally acquired IgG antibodies to a chimeric and non-chimeric recombinant species of Plasmodium vivax reticulocyte binding protein-1: lack of association with HLA-DRB1*/DQB1* in malaria exposed individuals from the Brazilian Amazon.

The development of modular constructs that include antigenic regions targeted by protective immune responses is an attractive approach for subunit vaccine development. However, a main concern of using these vaccine platforms is how to preserve the antigenic identity of conformational B cell epitopes. In the present study we evaluated naturally acquired antibody responses to a chimeric protein engineered to contain a previously defined immunodominant domain of the Plasmodium vivax reticulocyte binding protein-1 located between amino acid positions K435-I777. The construct also includes three regions of the cognate protein (F571-D587, I1745-S1786 and L2235-E2263) predicted to contain MHC class II promiscuous T cell epitopes. Plasma samples from 253 naturally exposed individuals were tested against this chimeric protein named PvRMC-RBP1 and a control protein that includes the native sequence PvRBP123-751 in comparative experiments to study the frequency of total IgG and IgG subclass reactivity. HLA-DRB1 and HLA-DQB1 allelic groups were typed by PCR-SSO to evaluate the association between major HLA class II alleles and antibody responses. We found IgG antibodies that recognized the chimeric PvRMC-RBP1 and the PvRBP123-751 in 47.1% and 60% of the studied population, respectively. Moreover, the reactivity index against both proteins were comparable and associated with time of exposure (p<0.0001) and number of previous malaria episodes (p<0.005). IgG subclass profile showed a predominance of cytophilic IgG1 over other subclasses against both proteins tested. Collectively these studies suggest that the chimeric PvRMC-RBP1 protein retained antigenic determinants in the PvRBP1435–777 native sequence. Although 52.9% of the population did not present detectable titers of antibodies to PvRMC-RBP1, genetic restriction to this chimeric protein does not seem to occur, since no association was observed between the HLA-DRB1* or HLA-DQB1* alleles and the antibody responses. This experimental evidence strongly suggests that the identity of the conformational B cell epitopes is preserved in the chimeric protein.

Comparative transcriptomics and metabolomics in a rhesus macaque drug administration study

We describe a multi-omic approach to understanding the effects that the anti-malarial drug pyrimethamine has on immune physiology in rhesus macaques (Macaca mulatta). Whole blood and bone marrow (BM) RNA-Seq and plasma metabolome profiles (each with over 15,000 features) have been generated for five naïve individuals at up to seven timepoints before, during and after three rounds of drug administration. Linear modeling and Bayesian network analyses are both considered, alongside investigations of the impact of statistical modeling strategies on biological inference. Individual macaques were found to be a major source of variance for both omic data types, and factoring individuals into subsequent modeling increases power to detect temporal effects. A major component of the whole blood transcriptome follows the BM with a time-delay, while other components of variation are unique to each compartment. We demonstrate that pyrimethamine administration does impact both compartments throughout the experiment, but very limited perturbation of transcript or metabolite abundance was observed following each round of drug exposure. New insights into the mode of action of the drug are presented in the context of pyrimethamines predicted effect on suppression of cell division and metabolism in the immune system.

A Plasmodium Promiscuous T Cell Epitope Delivered within the Ad5 Hexon Protein Enhances the Protective Efficacy of a Protein Based Malaria Vaccine

A malaria vaccine is a public health priority. In order to produce an effective vaccine, a multistage approach targeting both the blood and the liver stage infection is desirable. The vaccine candidates also need to induce balanced immune responses including antibodies, CD4+ and CD8+ T cells. Protein-based subunit vaccines like RTS,S are able to induce strong antibody response but poor cellular reactivity. Adenoviral vectors have been effective inducing protective CD8+ T cell responses in several models including malaria; nonetheless this vaccine platform exhibits a limited induction of humoral immune responses. Two approaches have been used to improve the humoral immunogenicity of recombinant adenovirus vectors, the use of heterologous prime-boost regimens with recombinant proteins or the genetic modification of the hypervariable regions (HVR) of the capsid protein hexon to express B cell epitopes of interest. In this study, we describe the development of capsid modified Ad5 vectors that express a promiscuous Plasmodium yoelii T helper epitope denominated PyT53 within the hexon HVR2 region. Several regimens were tested in mice to determine the relevance of the hexon modification in enhancing protective immune responses induced by the previously described protein-based multi-stage experimental vaccine PyCMP. A heterologous prime-boost immunization regime that combines a hexon modified vector with transgenic expression of PyCMP followed by protein immunizations resulted in the induction of robust antibody and cellular immune responses in comparison to a similar regimen that includes a vector with unmodified hexon. These differences in immunogenicity translated into a better protective efficacy against both the hepatic and red blood cell stages of P. yoelii. To our knowledge, this is the first time that a hexon modification is used to deliver a promiscuous T cell epitope. Our data support the use of such modification to enhance the immunogenicity and protective efficacy of adenoviral based malaria vaccines.

A Recombinant Chimeric Ad5/3 Vector Expressing a Multistage Plasmodium Antigen Induces Protective Immunity in Mice Using Heterologous Prime-Boost Immunization Regimens

An ideal malaria vaccine should target several stages of the parasite life cycle and induce antiparasite and antidisease immunity. We have reported a Plasmodium yoelii chimeric multistage recombinant protein (P. yoelii linear peptide chimera/recombinant modular chimera), engineered to express several autologous T cell epitopes and sequences derived from the circumsporozoite protein and the merozoite surface protein 1. This chimeric protein elicits protective immunity, mediated by CD4+ T cells and neutralizing Abs. However, experimental evidence, from pre-erythrocytic vaccine candidates and irradiated sporozoites, has shown that CD8+ T cells play a significant role in protection. Recombinant viral vectors have been used as a vaccine platform to elicit effective CD8+ T cell responses. The human adenovirus (Ad) serotype 5 has been tested in malaria vaccine clinical trials with excellent safety profile. Nevertheless, a major concern for the use of Ad5 is the high prevalence of anti-vector neutralizing Abs in humans, hampering its immunogenicity. To minimize the impact of anti-vector pre-existing immunity, we developed a chimeric Ad5/3 vector in which the knob region of Ad5 was replaced with that of Ad3, conferring partial resistance to anti-Ad5 neutralizing Abs. Furthermore, we implemented heterologous Ad/protein immunization regimens that include a single immunization with recombinant Ad vectors. Our data show that immunization with the recombinant Ad5/3 vector induces protective efficacy indistinguishable from that elicited by Ad5. Our study also demonstrates that the dose of the Ad vectors has an impact on the memory profile and protective efficacy. The results support further studies with Ad5/3 for malaria vaccine development.

Induction of Multifunctional Broadly Reactive T Cell Responses by a Plasmodium vivax Circumsporozoite Protein Recombinant Chimera

ABSTRACT Plasmodium vivax is the most widespread species of Plasmodium, causing up to 50% of the malaria cases occurring outside sub-Saharan Africa. An effective vaccine is essential for successful control and potential eradication. A well-characterized vaccine candidate is the circumsporozoite protein (CSP). Preclinical and clinical trials have shown that both antibodies and cellular immune responses have been correlated with protection induced by immunization with CSP. On the basis of our reported approach of developing chimeric Plasmodium yoelii proteins to enhance protective efficacy, we designed PvRMC-CSP, a recombinant chimeric protein based on the P. vivax CSP (PvCSP). In this engineered protein, regions of the PvCSP predicted to contain human T cell epitopes were genetically fused to an immunodominant B cell epitope derived from the N-terminal region I and to repeat sequences representing the two types of PvCSP repeats. The chimeric protein was expressed in soluble form with high yield. As the immune response to PvCSP has been reported to be genetically restricted in the murine model, we tested the immunogenicity of PvRMC-CSP in groups of six inbred strains of mice. PvRMC-CSP was able to induce robust antibody responses in all the mouse strains tested. Synthetic peptides representing the allelic forms of the P. vivax CSP were also recognized to a similar extent regardless of the mouse strain. Furthermore, the immunization regimen induced high frequencies of multifunctional CD4+ and CD8+ PvRMC-CSP-specific T cells. The depth and breadth of the immune responses elicited suggest that immunization with PvRMC-CSP can circumvent the genetic restriction of the immune response to P. vivax CSP. Interestingly, PvRMC-CSP was also recognized by naturally acquired antibodies from individuals living in areas where malaria is endemic. These features make PvRMC-CSP a promising vaccine candidate for further development.

Polymeric linear Peptide chimeric vaccine-induced antimalaria immunity is associated with enhanced in vitro antigen loading.

ABSTRACT Immunization of mice with Plasmodium berghei or Plasmodium yoelii synthetic linear peptide chimeras (LPCs) based on the circumsporozoite protein protects against experimental challenge with viable sporozoites. The immunogenicity of LPCs is significantly enhanced by spontaneous polymerization. To better understand the antigenic properties of polymeric antimalarial peptides, we studied the immune responses elicited in mice immunized with a polymer or a monomer of a linear peptide construct specific for P. yoelii and compared the responses of antigen-presenting cells following incubation with both peptide species. Efficient uptake of the polymeric peptide in vitro resulted in higher expression of the coactivation markers CD80, CD40, and CD70 on dendritic cells and higher proinflammatory cytokine production than with the monomeric peptide. Macropinocytosis seems to be the main route used by polymeric peptides internalized by antigen-presenting cells. Spontaneous polymerization of synthetic antimalarial-peptide constructs to target professional antigen-presenting cells shows promise for simple delivery of subunit malaria vaccines.