An unexpected protective role of low-affinity allergen-specific IgG through the inhibitory receptor FcγRIIb.

BACKGROUND: Induction of allergen-specific IgG antibodies is a critical parameter for successful allergen-specific immunotherapy. IgG antibodies can inhibit IgE-mediated mast cell activation through direct allergen neutralization or through the inhibitory receptor FcγRIIb. The affinity of IgE antibodies to the allergen has been shown to be critical for cellular activation. OBJECTIVE: Here we addressed the question of affinity thresholds of allergen-specific IgG antibodies for inhibition of mast cell activation using 2 different mAbs against the major cat allergen Fel d 1 both in vitro and in vivo in mice. METHODS: Sequences of the 2 high-affinity mAbs were back-mutated to germline, resulting in low-affinity (10-7 mol/L) antibodies of the exact same specificity. RESULTS: Using these newly generated recombinant antibodies, we demonstrate that low-affinity antibodies are still able to inhibit mast cell activation through FcγRIIb but do not neutralize the allergen. CONCLUSION: Antibody affinity dictates the mechanism of mast cell inhibition, and IgG antibodies triggering the inhibitory FcγRIIb pathway can show a broader cross-reactivity pattern than previously thought. This indicates that allergen-specific immunotherapy generates a larger protective umbrella of inhibitory IgG antibodies than previously appreciated.

The CD14+CD16+ inflammatory monocyte subset displays increased mitochondrial activity and effector function during acute Plasmodium vivax malaria.

Infection with Plasmodium vivax results in strong activation of monocytes, which are important components of both the systemic inflammatory response and parasite control. The overall goal of this study was to define the role of monocytes during P. vivax malaria. Here, we demonstrate that P. vivax-infected patients display significant increase in circulating monocytes, which were defined as CD14(+)CD16- (classical), CD14(+)CD16(+) (inflammatory), and CD14loCD16(+) (patrolling) cells. While the classical and inflammatory monocytes were found to be the primary source of pro-inflammatory cytokines, the CD16(+) cells, in particular the CD14(+)CD16(+) monocytes, expressed the highest levels of activation markers, which included chemokine receptors and adhesion molecules. Morphologically, CD14(+) were distinguished from CD14lo monocytes by displaying larger and more active mitochondria. CD14(+)CD16(+) monocytes were more efficient in phagocytizing P. vivax-infected reticulocytes, which induced them to produce high levels of intracellular TNF-α and reactive oxygen species. Importantly, antibodies specific for ICAM-1, PECAM-1 or LFA-1 efficiently blocked the phagocytosis of infected reticulocytes by monocytes. Hence, our results provide key information on the mechanism by which CD14(+)CD16(+) cells control parasite burden, supporting the hypothesis that they play a role in resistance to P. vivax infection.

Induction of Inhibitory Receptors on T Cells During Plasmodium vivax Malaria Impairs Cytokine Production.

The function and regulation of the immune response triggered during malaria is complex and poorly understood, and there is a particular paucity of studies conducted in humans infected with Plasmodium vivax. While it has been proposed that T-cell-effector responses are crucial for protection against blood-stage malaria in mice, the mechanisms behind this in humans remain poorly understood. Experimental models of malaria have shown that the regulatory molecules, cytotoxic T-lymphocyte attenuator-4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), and programmed death-1 (PD-1) are involved in the functional impairment of T cells during infection. Our goal was to define the role of these molecules during P. vivax malaria. We demonstrate that infection triggers the expression of regulatory molecules on T cells. The pattern of expression differs in CD4(+) and CD8(+) T cells. Higher frequencies of CD4(+) express more than 1 regulatory molecule compared to CD8(+) T cells. Moreover, lower proportions of CD4(+) T cells coexpress regulatory molecules, but are still able to proliferate. Importantly, simultaneously blockade of the CLTA-4, PD-1, and T-cell immunoglobulin and mucin-3 signaling restores the cytokine production by antigen-specific cells. These data support the hypothesis that upregulation of inhibitory receptors on T cells during P. vivax malaria impairs parasite-specific T-cell effector function.

Drug-cured experimental Trypanosoma cruzi infections confer long-lasting and cross-strain protection.

BACKGROUND: The long term and complex nature of Chagas disease in humans has restricted studies on vaccine feasibility. Animal models also have limitations due to technical difficulties in monitoring the extremely low parasite burden that is characteristic of chronic stage infections. Advances in imaging technology offer alternative approaches that circumvent these problems. Here, we describe the use of highly sensitive whole body in vivo imaging to assess the efficacy of recombinant viral vector vaccines and benznidazole-cured infections to protect mice from challenge with Trypanosoma cruzi. METHODOLOGY/PRINCIPAL FINDINGS: Mice were infected with T. cruzi strains modified to express a red-shifted luciferase reporter. Using bioluminescence imaging, we assessed the degree of immunity to re-infection conferred after benznidazole-cure. Those infected for 14 days or more, prior to the onset of benznidazole treatment, were highly protected from challenge with both homologous and heterologous strains. There was a >99% reduction in parasite burden, with parasites frequently undetectable after homologous challenge. This level of protection was considerably greater than that achieved with recombinant vaccines. It was also independent of the route of infection or size of the challenge inoculum, and was long-lasting, with no significant diminution in immunity after almost a year. When the primary infection was benznidazole-treated after 4 days (before completion of the first cycle of intracellular infection), the degree of protection was much reduced, an outcome associated with a minimal T. cruzi-specific IFN-γ+ T cell response. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that a protective Chagas disease vaccine must have the ability to eliminate parasites before they reach organs/tissues, such as the GI tract, where once established, they become largely refractory to the induced immune response.

Evaluation of Chimpanzee Adenovirus and MVA Expressing TRAP and CSP from Plasmodium cynomolgi to Prevent Malaria Relapse in Nonhuman Primates.

Plasmodium vivax is the world's most widely distributed human malaria parasite, with over 2.8 billion people at risk in Asia, the Americas, and Africa. The 80-90% new P. vivax malaria infections are due to relapses which suggest that a vaccine with high efficacy against relapses by prevention of hypnozoite formation could lead to a significant reduction in the prevalence of P. vivax infections. Here, we describe the development of new recombinant ChAdOx1 and MVA vectors expressing P. cynomolgi Thrombospondin Related Adhesive Protein (PcTRAP) and the circumsporozoite protein (PcCSP). Both were shown to be immunogenic in mice prior to their assessment in rhesus macaques. We confirmed good vaccine-induced humoral and cellular responses after prime-boost vaccination in rhesus macaques prior to sporozoite challenge. Results indicate that there were no significant differences between mock-control and vaccinated animals after challenge, in terms of protective efficacy measured as the time taken to 1st patency, or as number of relapses. This suggests that under the conditions tested, the vaccination with PcTRAP and PcCSP using ChAdOx1 or MVA vaccine platforms do not protect against pre-erythrocytic malaria or relapses despite good immunogenicity induced by the viral-vectored vaccines.

RNA and Toll-Like Receptor 7 License the Generation of Superior Secondary Plasma Cells at Multiple Levels in a B Cell Intrinsic Fashion.

Secondary plasma cells (PCs) originate from memory B cells and produce increased levels of antibodies with higher affinity compared to PCs generated during primary responses. Here we demonstrate that virus-like particles (VLPs) only induce secondary PCs in the presence of toll-like receptor (TLR) 7 and if they are loaded with RNA. Furthermore, adoptive transfer experiments demonstrate that RNA and TLR7 signaling are required for secondary PC generation, both at the level of memory B cell as well as PC differentiation. TLR7-signaling occurred in a B cell intrinsic manner as TLR7-deficient B cells in an otherwise TLR7-competent environment failed to differentiate into secondary PCs. Therefore, RNA inside VLPs is essential for the generation of memory B cells, which are competent to differentiate to secondary PCs and for the differentiation of secondary PCs themselves. While we have not tested all other TLR or non-TLR adjuvants with our VLPs, these data have obvious implications for vaccine design, as RNA packaged into VLPs is a simple way to enhance induction of memory B cells capable of generating secondary PCs.

Early Transcriptional Signature in Dendritic Cells and the Induction of Protective T Cell Responses Upon Immunization With VLPs Containing TLR Ligands-A Role for CCL2.

Inducing T cell responses by therapeutic vaccination requires appropriate activation of antigen presenting cells (APCs). The use of virus-like particles (VLPs) containing Toll-like receptor (TLR) ligands has demonstrated remarkable potential in activating APCs and modulating the immune response both for prophylactic vaccines as well as immunotherapy. Here, we employed VLPs associated to TLR ligands as tools to modulate cytotoxic response mediated by CD8+ T cells and provide further insight in the development of T cell-based immunotherapy. We have investigated the in vivo transcriptional signature in dendritic cells (DCs) from mice immunized with VLPs containing distinct classes of nucleic acid and correlated the expression patterns with the efficiency of induced T cell responses. We identified key pathways activated in DCs that are involved in the appropriated induction of T cell responses and show evidence for the modulatory effect of CCL2 in CD8+ T cells responses. These insights shed light on immune networks that are pivotal for the induction of potent cytotoxic T cell responses and identify key genes for appropriate DC activation and subsequent modulation of the adaptive immune response.

Pattern of humoral immune response to Plasmodium falciparum blood stages in individuals presenting different clinical expressions of malaria.

BACKGROUND: The development of protective immunity against malaria is slow and to be maintained, it requires exposure to multiple antigenic variants of malaria parasites and age-associated maturation of the immune system. Evidence that the protective immunity is associated with different classes and subclasses of antibodies reveals the importance of considering the quality of the response. In this study, we have evaluated the humoral immune response against Plasmodium falciparum blood stages of individuals naturally exposed to malaria who live in endemic areas of Brazil in order to assess the prevalence of different specific isotypes and their association with different malaria clinical expressions. METHODS: Different isotypes against P. falciparum blood stages, IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgE and IgA, were determined by ELISA. The results were based on the analysis of different clinical expressions of malaria (complicated, uncomplicated and asymptomatic) and factors related to prior malaria exposure such as age and the number of previous clinical malaria attacks. The occurrence of the H131 polymorphism of the FcgammaIIA receptor was also investigated in part of the studied population. RESULTS: The highest levels of IgG, IgG1, IgG2 and IgG3 antibodies were observed in individuals with asymptomatic and uncomplicated malaria, while highest levels of IgG4, IgE and IgM antibodies were predominant among individuals with complicated malaria. Individuals reporting more than five previous clinical malaria attacks presented a predominance of IgG1, IgG2 and IgG3 antibodies, while IgM, IgA and IgE antibodies predominated among individuals reporting five or less previous clinical malaria attacks. Among individuals with uncomplicated and asymptomatic malaria, there was a predominance of high-avidity IgG, IgG1, IgG2 antibodies and low-avidity IgG3 antibodies. The H131 polymorphism was found in 44.4% of the individuals, and the highest IgG2 levels were observed among asymptomatic individuals with this allele, suggesting the protective role of IgG2 in this population. CONCLUSION: Together, the results suggest a differential regulation in the anti-P. falciparum antibody pattern in different clinical expressions of malaria and showed that even in unstable transmission areas, protective immunity against malaria can be observed, when the appropriated antibodies are produced.

Cross sectional study reveals a high percentage of asymptomatic Plasmodium vivax infection in the Amazon Rio Negro area, Brazil.

A parasitological, clinical, serological and molecular cross-sectional study carried out in a highly endemic malaria area of Rio Negro in the Amazon State, Brazil, revealed a high prevalence of asymptomatic Plasmodium vivax infection. A total of 109 persons from 25 families were studied in five villages. Ninety-nine inhabitants (90.8%) had at least one previous episode of malaria. Serology showed 85.7% and 46.9% of positivity when P. falciparum antigens and P. vivax MSP-1, respectively, were used. Twenty blood samples were PCR positive for P. vivax (20.4%) and no P. falciparum infection was evidenced by this technique. No individual presenting positive PCR reaction had clinical malaria during the survey neither in the six months before nor after, confirming that they were cases of asymptomatic infection. Only one 12 year old girl presented a positive thick blood smear for P. vivax. This is the first description of asymptomatic Plasmodium infection in this area studied.

Virus-Like Particle (VLP) Plus Microcrystalline Tyrosine (MCT) Adjuvants Enhance Vaccine Efficacy Improving T and B Cell Immunogenicity and Protection against Plasmodium berghei/vivax.

Vaccination is the most effective prophylactic tool against infectious diseases. Despite continued efforts to control malaria, the disease still generally represents a significant unmet medical need. Microcrystalline tyrosine (MCT) is a well described depot used in licensed allergy immunotherapy products and in clinical development. However, its proof of concept in prophylactic vaccines has only recently been explored. MCT has never been used in combination with virus-like particles (VLPs), which are considered to be one of the most potent inducers of cellular and humoral immune responses in mice and humans. In the current study we assessed the potential of MCT to serve as an adjuvant in the development of a vaccine against malaria either alone or combined with VLP using Plasmodium vivax thrombospondin-related adhesive protein (TRAP) as a target antigen. We chemically coupled PvTRAP to VLPs derived from the cucumber mosaic virus fused to a universal T-cell epitope of the tetanus toxin (CMVtt), formulated with MCT and compared the induced immune responses to PvTRAP formulated in PBS or Alum. The protective capacity of the various formulations was assessed using Plasmodium berghei expressing PvTRAP. All vaccine formulations using adjuvants and/or VLP increased humoral immunogenicity for PvTRAP compared to the antigen alone. The most proficient responder was the group of mice immunized with the vaccine formulated with PvTRAP-VLP + MCT. The VLP-based vaccine formulated in MCT also induced the strongest T cell response and conferred best protection against challenge with recombinant Plasmodium berghei. Thus, the combination of VLP with MCT may take advantage of the properties of each component and appears to be an alternative biodegradable depot adjuvant for development of novel prophylactic vaccines.

Variants in the toll-like receptor signaling pathway and clinical outcomes of malaria.

BACKGROUND: Malaria is one of the most significant infectious diseases in the world and is responsible for a large proportion of infant deaths. Toll-like receptors (TLRs), key components of innate immunity, are central to countering infection. Variants in the TLR-signaling pathway are associated with susceptibility to infectious diseases. METHODS: We genotyped single nucleotide polymorphisms (SNPs) of the genes associated with the TLR-signaling pathway in patients with mild malaria and individuals with asymptomatic Plasmodium infections by means of polymerase chain reaction. RESULTS: Genotype distributions for the TLR-1 I602S differed significantly between patients with mild malaria and persons with asymptomatic infection. The TLR-1 602S allele was associated with an odds ratio (OR) of 2.2 (P= .003; P(corrected)= .015) for malaria among patients with mild malaria due to any Plasmodium species and 2.1 (P= .015; P(corrected)= .75) among patients with mild malaria due to Plasmodium falciparum only. The TLR-6 S249P SNP showed an excess of homozygotes for the TLR-6 249P allele in asymptomatic persons, compared with patients with mild malaria due to any Plasmodium species (OR 2.1; 95% confidence interval [CI], 1.1- 4.2; P= .01; P(corrected)= .05), suggesting that the TLR-6 249S allele may be a risk factor for malaria (OR, 2.0; 95% CI, 1.1-3.7; P=0.01; P(corrected)= .05). The TLR-9 -1486C allele showed a strong association with high parasitemia (P< .001). CONCLUSIONS: Our findings indicate that the TLR-1 and TLR-6 variants are significantly associated with mild malaria, whereas the TLR-9-1486C/T variants are associated with high parasitemia. These discoveries may bring additional understanding to the pathogenesis of malaria.

Differential recognition of Plasmodium falciparum merozoite surface protein 2 variants by antibodies from malaria patients in Brazil.

Four variants of merozoite surface protein 2 (MSP-2) of Plasmodium falciparum were used in serology to examine whether changes in repeat units affect its recognition by antibodies during infection with parasites of known MSP-2 types. The results indicate that variation in MSP-2 repeats may represent a mechanism for parasite immune evasion.

Cross sectional study reveals a high percentage of asymptomatic Plasmodium vivax infection in the Amazon Rio Negro area, Brazil

A parasitological, clinical, serological and molecular cross-sectional study carried out in a highly endemic malaria area of Rio Negro in the Amazon State, Brazil, revealed a high prevalence of asymptomatic Plasmodium vivax infection. A total of 109 persons from 25 families were studied in five villages. Ninety-nine inhabitants (90.8 percent) had at least one previous episode of malaria. Serology showed 85.7 percent and 46.9 percent of positivity when P. falciparum antigens and P. vivax MSP-1, respectively, were used. Twenty blood samples were PCR positive for P. vivax (20.4 percent) and no P. falciparum infection was evidenced by this technique. No individual presenting positive PCR reaction had clinical malaria during the survey neither in the six months before nor after, confirming that they were cases of asymptomatic infection. Only one 12 year old girl presented a positive thick blood smear for P. vivax. This is the first description of asymptomatic Plasmodium infection in this area studied.

Um estudo seccional parasitológico, clínico, sorológico e molecular, realizado em uma área altamente endêmica para malária, no Rio Negro, Estado do Amazonas, revela alta prevalência de infecção assintomática por Plasmodium vivax. Um total de 109 pessoas de 25 famílias residentes em cinco comunidades do Rio Padauiri, afluente do Rio Negro, foram estudadas. Noventa por cento dos habitantes (90,8 por cento) tinham tido pelo menos um episodio prévio de malária. A sorologia mostrou 85,7 por cento e 46,9 por cento de positividade quando antígenos de P. falciparum e P. vivax MSP-1, foram respectivamente usados. Vinte amostras de sangue submetidas ao PCR foram positivas para P. vivax (20,4 por cento), entretanto, nenhuma foi positiva para o P. falciparum por esta técnica. Nenhum paciente com PCR positivo durante o inquérito e seis meses antes ou depois teve manifestações clínicas de malária, portanto, podemos afirmar que eram assintomáticos. Somente uma criança de 12 anos de idade teve gota espessa positiva para P. vivax. Esta é a primeira descrição de infecção assintomática por Plasmodium na área estudada.