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Abstract A safe and effective vaccine with long-term protection against SARS-CoV-2 variants of concern (VOCs) is a global health priority. Here, we develop lipid nanoparticles (LNPs) to provide safe and effective delivery of plasmid DNA (pDNA) and show protection against VOCs in female small animal models. Using a library of LNPs encapsulating unique barcoded DNA (b-DNA), we screen for b-DNA delivery after intramuscular administration. The top-performing LNPs are further tested for their capacity of pDNA uptake in antigen-presenting cells in vitro. The lead LNP is used to encapsulate pDNA encoding the HexaPro version of SARS-CoV-2 spike (LNP-HPS) and immunogenicity and protection is tested in vivo. LNP-HPS elicit a robust protective effect against SARS-CoV-2 Gamma (P.1), correlating with reduced lethality, decreased viral load in the lungs and reduced lung damage. LNP-HPS induce potent humoral and T cell responses against P.1, and generate high levels of neutralizing antibodies against P.1 and Omicron (B.1.1.529). Our findings indicate that the protective efficacy and immunogenicity elicited by LNP-HPS are comparable to those achieved by the approved COVID-19 vaccine from Biontech/Pfizer in animal models. Together, these findings suggest that LNP-HPS hold great promise as a vaccine candidate against VOCs.

Abstract Kupffer cells (KCs) are self-maintained tissue-resident macrophages that line liver sinusoids and play an important role on host defense. It has been demonstrated that upon infection or intense liver inflammation, KCs might be severely depleted and replaced by immature monocytic cells; however, the mechanisms of cell death and the alterations on liver immunity against infections deserves further investigation. We explored the impact of acute Plasmodium infection on KC biology and on the hepatic immune response against secondary infections. Similar to patients, infection with Plasmodium chabaudi induced acute liver damage as determined by serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) elevation. This was associated with accumulation of hemozoin, increased of proinflammatory response and impaired bacterial and viral clearance, which led to pathogen spread to other organs. In line with this, mice infected with Plasmodium had enhanced mortality during secondary infections, which was associated with increased production of mitochondrial superoxide, lipid peroxidation and increased free iron within KCs—hallmarks of cell death by ferroptosis. Therefore, we revealed that accumulation of iron with KCs, triggered by uptake of circulating hemozoin, is a novel mechanism of macrophage depletion and liver inflammation during malaria, providing novel insights on host susceptibility to secondary infections. Malaria can cause severe liver damage, along with depletion of liver macrophages, which can predispose individuals to secondary infections and enhance the chances of death.

BackgroundPlasmodium spp. infection triggers the production of inflammatory cytokines that are essential for parasite control, and conversely responsible for symptoms of malaria. Monocytes play a role in host defense against Plasmodium vivax infection and represent the main source of inflammatory cytokines and reactive oxygen species. The anti-inflammatory cytokine IL-10 is a key regulator preventing exacerbated inflammatory responses. Studies suggested that different clinical presentations of malaria are strongly associated with an imbalance in the production of inflammatory and anti-inflammatory cytokines.MethodsA convenience sampling of peripheral blood mononuclear cells from Plasmodium vivax-infected patients and healthy donors were tested for the characterization of cytokine and adenosine production and the expression of ectonucleotidases and purinergic receptors.ResultsHere we show that despite a strong inflammatory response, monocytes also bear a modulatory role during malaria. High levels of IL-10 are produced during P. vivax infection and its production can be triggered in monocytes by P. vivax-infected reticulocytes. Monocytes express high levels of ectonucleotidases, indicating their important role in extracellular ATP modulation and consequently in adenosine production. Plasmatic levels of adenosine are not altered in patients experiencing acute malaria; however, their monocyte subsets displayed an increased expression of P1 purinergic receptors. In addition, adenosine decreases Tumor Necrosis Factor production by monocytes, which was partially abolished with the blockage of the A2a receptor.ConclusionMonocytes have a dual role, attempting to control both the P. vivax infection and the inflammatory response. Purinergic receptor modulators emerge as an untapped approach to ameliorate clinical malaria.

There is a massive demand to identify alternative methods to detect new cases of COVID-19 as well as to investigate the epidemiology of the disease. In many countries, importation of commercial kits poses a significant impact on their testing capacity and increases the costs for the public health system. We have developed an ELISA to detect IgG antibodies against SARS-CoV-2 using a recombinant viral nucleocapsid (rN) protein expressed in E. coli. Using a total of 894 clinical samples we showed that the rN-ELISA was able to detect IgG antibodies against SARS-CoV-2 with high sensitivity (97.5%) and specificity (96.3%) when compared to a commercial antibody test. After three external validation studies, we showed that the test accuracy was higher than 90%. The rN-ELISA IgG kit constitutes a convenient and specific method for the large-scale determination of SARS-CoV-2 antibodies in human sera with high reliability.

Individuals with asymptomatic infection due to Plasmodium vivax are posited to be important reservoirs of malaria transmission in endemic regions. Here we studied a cohort of P. vivax malaria patients in a suburban area in the Brazilian Amazon. Overall 1,120 individuals were screened for P. vivax infection and 108 (9.6%) had parasitemia detected by qPCR but not by microscopy. Asymptomatic individuals had higher levels of antibodies against P. vivax and similar hematological and biochemical parameters compared to uninfected controls. Blood from asymptomatic individuals with very low parasitemia transmitted P. vivax to the main local vector, Nyssorhynchus darlingi. Lower mosquito infectivity rates were observed when blood from asymptomatic individuals was used in the membrane feeding assay. While blood from symptomatic patients infected 43.4% (199/458) of the mosquitoes, blood from asymptomatic infected 2.5% (43/1,719). However, several asymptomatic individuals maintained parasitemia for several weeks indicating their potential role as an infectious reservoir. These results suggest that asymptomatic individuals are an important source of malaria parasites and Science and Technology for Vaccines granted by Conselho Nacional de may contribute to the transmission of P. vivax in low-endemicity areas of malaria. Author summary Malaria still poses as one of the most important parasitic diseases in the world. The advance of molecular diagnosis brought to light the existence of asymptomatic infections, which may represent most of the infections in some areas. Importantly, the role of asymptomatic carriers in the natural history of malaria is not completely understood. Herein we describe the general characteristics of asymptomatic individuals infected with Plasmodium vivax, and provide evidence of their potential as parasitic reservoirs, even when molecular methods fail to detect the infection. Our findings reinforce the need for better diagnostic tests and open a new window of complexity to be considered in control programs.

Individuals with asymptomatic infection due to Plasmodium vivax are posited to be important reservoirs of malaria transmission in endemic regions. Here we studied a cohort of P. vivax malaria patients in a suburban area in the Brazilian Amazon. Overall 1,120 individuals were screened for P. vivax infection and 108 (9.6%) had parasitemia detected by qPCR but not by microscopy. Asymptomatic individuals had higher levels of antibodies against P. vivax and similar hematological and biochemical parameters compared to uninfected controls. Blood from asymptomatic individuals with very low parasitemia transmitted P. vivax to the main local vector, Nyssorhynchus darlingi. Lower mosquito infectivity rates were observed when blood from asymptomatic individuals was used in the membrane feeding assay. While blood from symptomatic patients infected 43.4% (199/458) of the mosquitoes, blood from asymptomatic infected 2.5% (43/1,719). However, several asymptomatic individuals maintained parasitemia for several weeks indicating their potential role as an infectious reservoir. These results suggest that asymptomatic individuals are an important source of malaria parasites and Science and Technology for Vaccines granted by Conselho Nacional de may contribute to the transmission of P. vivax in low-endemicity areas of malaria.

ABSTRACT Monocytes play an important role in the host defense against Plasmodium vivax as the main source of inflammatory cytokines and mitochondrial reactive oxygen species (mROS). Here, we show that monocyte metabolism is altered during human P. vivax malaria, with mitochondria playing a major function in this switch. The process involves a reprograming in which the cells increase glucose uptake and produce ATP via glycolysis instead of oxidative phosphorylation. P. vivax infection results in dysregulated mitochondrial gene expression and in altered membrane potential leading to mROS increase rather than ATP production. When monocytes were incubated with P. vivax-infected reticulocytes, mitochondria colocalized with phagolysosomes containing parasites representing an important source mROS. Importantly, the mitochondrial enzyme superoxide dismutase 2 (SOD2) is simultaneously induced in monocytes from malaria patients. Taken together, the monocyte metabolic reprograming with an increased mROS production may contribute to protective responses against P. vivax while triggering immunomodulatory mechanisms to circumvent tissue damage. IMPORTANCE Plasmodium vivax is the most widely distributed causative agent of human malaria. To achieve parasite control, the human immune system develops a substantial inflammatory response that is also responsible for the symptoms of the disease. Among the cells involved in this response, monocytes play an important role. Here, we show that monocyte metabolism is altered during malaria, with its mitochondria playing a major function in this switch. This change involves a reprograming process in which the cells increase glucose uptake and produce ATP via glycolysis instead of oxidative phosphorylation. The resulting altered mitochondrial membrane potential leads to an increase in mitochondrial reactive oxygen species rather than ATP. These data suggest that agents that change metabolism should be investigated and used with caution during malaria.

Summary. Human visceral leishmaniasis (VL) is a major public health problem worldwide, leading to significant mortality rates if not properly treated and controlled. Precise identification of infected patients is essential to establish treatment and control measures. Although several VL serological diagnosis advances have been accomplished lately, mainly using recombinant antigens and immunochromatographic tests (ICTs), improvements may still be achieved using multiepitope chimeric proteins in different test platforms. Here, we reported on the evaluation of ELISA and an ICT developed with a new chimeric protein, named DTL-4, based on repetitive antigenic sequences, including those present in the A2 protein. Methods. A total of 1028 sera samples were used for the development and validation of ELISA (321 samples from L. infantum-infected patients, 62 samples from VL/AIDS coinfected patients, 236 samples from patients infected with other diseases, and 409 samples from healthy donors). A total of 520 sera samples were used to develop and validate ICT (249 samples from L. infantum-infected patients, 46 samples from VL/AIDS coinfected patients, 40 samples from patients infected with other diseases, and 185 samples from healthy donors). Findings. Using the validation sera panels, DTL-4-based ELISA displayed an overall sensitivity of 94.61% (95% CI: 89.94-97.28), a specificity of 99.41% (95% CI: 96.39-99.99), and an accuracy of 97.02% (95% CI: 94.61-98.38), while for ICT, sensitivity, specificity, and accuracy values corresponded to 91.98% (95% CI: 86.65-95.39), 100.00% (95% CI: 96.30-100.00), and 95.14% (95% CI: 91.62-97.15), respectively. When testing sera samples from VL/AIDS coinfected patients, DTL-4-ELISA displayed a sensitivity of 77.42% (95% CI: 65.48-86.16), a specificity of 99.41% (95% CI: 96.39-99.99), and an accuracy of 93.51% (95% CI: 89.49%-96.10%), while for DTL-4-ICT, sensitivity was 73.91% (95% CI: 59.74-84.40), specificity was 90.63% (95% CI: 81.02-95.63), and accuracy was 82.00% (95% CI: 73.63-90.91). Conclusion. DTL-4 is a promising candidate antigen for serodiagnosis of VL patients, including those with VL/AIDS coinfection, when incorporated into ELISA or ICT test formats.

We describe here the development of an in-house enzyme linked immunosorbent assay (ELISA) for the diagnostic of Chikungunya virus (CHIKV) infections using a recombinant protein from CHIKV. The recombinant protein gene was designed based on 154 sequences and we used computational methods to predict its structure and antigenic potential. To confirm predictions, the gene coding for the recombinant CHIKV protein (rCHIKVp) was synthetized and expressed in prokaryotic system. Subsequently, the protein was purified by affinity chromatography and used as antigen in an indirect ELISA. We present data regarding the optimization of the recombinant antigen production and preparation of the ELISA to detect IgG against CHIKV in human sera. Keywords: Chikungunya virus, Diagnostics, ELISA, E2 Envelope protein, Recombinant protein

BACKGROUND Visceral Leishmaniasis (VL) is an infectious disease that is a significant cause of death among infants aged under 1 year and the elderly in Brazil. Serodiagnosis is a mainstay of VL elimination programs; however, it has significant limitations due to low accuracy. OBJECTIVE This study aimed to evaluate three recombinant Leishmania infantum proteins (rFc, rC9, and rA2) selected from previous proteomics and genomics analyses to develop enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests (ICT) for the serodiagnosis of human VL (HVL) and canine VL (CVL). METHODS A total of 186 human (70 L. infantum-infected symptomatic, 20 other disease-infected, and 96 healthy) and 185 canine (82 L. infantum-infected symptomatic, 27 L. infantum-infected asymptomatic, and 76 healthy) sera samples were used for antibody detection. FINDINGS Of the three proteins, rA2 (91.5% sensitivity and 87% specificity) and rC9 (95.7% sensitivity and 87.5% specificity) displayed the best performance in ELISA-HVL and ELISA-CVL, respectively. ICT-rA2 also displayed the best performance for HVL diagnosis (92.3% sensitivity and 88.0% specificity) and had high concordance with immunofluorescence antibody tests (IFAT), ELISA-rK39, IT-LEISH®, and ELISAEXT. ICT-rFc, ICT-rC9, and ICT-rA2 had sensitivities of 88.6%, 86.5%, and 87.0%, respectively, with specificity values of 84.0%, 92.0%, and 100%, respectively for CVL diagnosis. MAIN CONCLUSIONS The three antigens selected by us are promising candidates for VL diagnosis regardless of the test format, although the antigen combinations and test parameters may warrant further optimisation.

Chikungunya virus (CHIKV) is a mosquito-borne pathogen that causes a disease characterized by the acute onset of fever accompanied by arthralgia and intense joint pain. Clinical similarities and cocirculation of this and other arboviruses in many tropical countries highlight the necessity for efficient and accessible diagnostic tools. CHIKV envelope proteins are highly conserved among alphaviruses and, particularly, the envelope 2 glycoprotein (CHIKV-E2) appears to be immunodominant and has a considerable serodiagnosis potential. Here, we investigate how glycosylation of CHIKV-E2 affects antigen/antibody interaction and how this affects the performance of CHIKV-E2-based Indirect ELISA tests. We compare two CHIKV-E2 recombinant antigens produced in different expression systems: prokaryotic-versus eukaryotic-made recombinant proteins. CHIKV-E2 antigens are expressed either in E. coli BL21(DE3)—a prokaryotic system unable to produce post-translational modifications—or in HEK-293T mammalian cells—a eukaryotic system able to add post-translational modifications, including glycosylation sites. Both prokaryotic and eukaryotic recombinant CHIKV-E2 react strongly to anti-CHIKV IgG antibodies, showing accuracy levels that are higher than 90%. However, the glycan-added viral antigen presents better sensitivity and specificity (85 and 98%) than the non-glycosylated antigen (81 and 71%, respectively) in anti-CHIKV IgM ELISA assays.

Although the importance of humoral immunity to malaria has been established, factors that control antibody production are poorly understood. Follicular helper T cells (Tfh cells) are pivotal for generating high-affinity, long-lived antibody responses. While it has been proposed that expansion of antigen-specific Tfh cells, interleukin (IL) 21 production and robust germinal center formation are associated with protection against malaria in mice, whether Tfh cells are found during Plasmodium vivax (P. vivax) infection and if they play a role during disease remains unknown. Our goal was to define the role of Tfh cells during P. vivax malaria. We demonstrate that P. vivax infection triggers IL-21 production and an increase in Tfh cells (PD-1+ICOS+CXCR5+CD45RO+CD4+CD3+). As expected, FACS-sorted Tfh cells, the primary source of IL-21, induced immunoglobulin production by purified naïve B cells. Furthermore, we found that P. vivax infection alters the B cell compartment and these alterations were dependent on the number of previous infections. First exposure leads to increased proportions of activated and atypical memory B cells and decreased frequencies of classical memory B cells, whereas patients that experienced multiple episodes displayed lower proportions of atypical B cells and higher frequencies of classical memory B cells. Despite the limited sample size, but consistent with the latter finding, the data suggest that patients who had more than five infections harbored more Tfh cells and produce more specific antibodies. P. vivax infection triggers IL-21 production by Tfh that impact B cell responses in humans.

The objective of the present study was to investigate the histopathological alterations and detection of parasites that occur in the eyes of dogs naturally infected with Leishmania chagasi. The eyes of 25 dogs with a positive parasitological and serological exam for L. chagasi were submitted to routine ophthalmic examination. Fragments were obtained from the eyeball and were analyzed by immunohistochemistry and histopathology. Among the structures studied, the conjunctiva of the third eyelid was the tissue most frequently stained for L. chagasi. Histopathological analysis revealed a predominantly mononuclear inflammatory infiltrate whose intensity ranged from discrete to intense in the various ocular structures. Vascular congestion and perivasculitis were also observed. The histopathological alterations that occur in the eyeball of dogs with visceral leishmaniasis might be related to the presence of antibodies.O objetivo deste trabalho foi estudar as alterações histopatológicas e a detecção do parasito nos olhos de cães naturalmente infectados por Leishmania chagasi. Utilizaram-se os olhos de 25 cães com exame parasitológico e sorológico positivo para Leishmania chagasi. Os animais foram submetidos à semiotécnica oftálmica rotineira. Fragmentos do globo ocular foram coletados e destinados à imunoistoquímica e histopatologia. Entre as estruturas avaliadas, a conjuntiva da terceira pálpebra foi a que mais demonstrou imunomarcação da Leishmania chagasi. Na avaliação histopatológica, foi observado predominantemente infiltrado inflamatório mononuclear, variando de discreto a intenso nas diversas estruturas oculares. Congestão vascular e perivasculite também foram observadas. As alterações histopatológicas do bulbo do olho em cães com leishmaniose visceral podem estar relacionadas à presença do anticorpo.

BACKGROUND: American tegumentary leishmaniasis (ATL) is endemic in Latin America, where Brazil has over 27 thousand cases per year. The aim of the present study was to develop an immunohistochemical method (IHC) for ATL diagnosis. For this purpose, we used serum from a dog naturally infected with Leishmania (L) infantum (canine hyperimmune serum) as the primary antibody, followed by a detection system with a secondary biotinylated antibody. METHODOLOGY: Skin samples were obtained from 73 patients in an endemic area of Caratinga, Minas Gerais (MG) State, Brazil all testing positive for ATL with the Montenegro skin test, microscopy, and PCR. Canine hyperimmune serum of a dog naturally infected with Leishmania (L.) infantum was employed as a primary antibody in an immunohistochemical diagnostic method using streptavidin-biotin peroxidase. To assess the specificity of this reaction, IHC assays employing two monoclonal antibodies were carried out. As the polymer-based technology is less time-consuming and labor intensive than the IHC labeled streptavidin-biotin peroxidase method, we compared the two methods for all samples. RESULTS: The IHC method detected ATL in 67 of the 73 cases (91.8%). Immunolabeled parasites were primarily detected inside macrophages either in the superficial or the deep dermis. Detection was facilitated by the high contrast staining of amastigotes (dark brown) against the light blue background. A lower detection rate (71.2%) was observed with the both of the monoclonal Leishmania antibodies compared to the canine hyperimmune serum. This may have been due to a non-specific background staining observed in all histological samples rendering positive detection more difficult. The higher efficacy of the canine hyperimmune serum in the IHC method was confirmed by the method using streptavidin-biotin peroxidase as well as that with the polymer-based technology (biotin-avidin-free system). CONCLUSIONS: The data are encouraging with regard to validating IHC as a standard alternative method for ATL diagnosis.

The aim of the present study was to investigate TLR2 expression in peripheral blood monocytes from dogs naturally infected with Leishmania (Leishmania) infantum to determine whether it correlates with CD11b/CD18 (CR3) expression, and to evaluate the potential of dogs as sources of infection using phlebotomine xenodiagnosis. Forty eight dogs were serologically diagnosed with L. infantum infection by indirect immunofluorescence antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA). Parasitological exams from bone-marrow aspirates were positive by PCR analysis. All dogs were clinical defined as symptomatic. Ear skin tissue samples were obtained for immunohistochemistry (IHQ) analysis. The potential of these dogs as a source of infection using phlebotomine xenodiagnosis (XENO) was evaluated. Flow cytometry was carried out on peripheral blood mononuclear cells using superficial receptors including CD14, CD11b, TLR2 and MHCII. IHQ ear skin tissue parasite load and XENO where done where we found a strict correlation (r = 0.5373). Dogs with higher expression of MFI of CD11b inside CD14 monocytes were represented by dogs without parasite ear tissue load that were unable to infect phlebotomines (IHQ⁻/XENO⁻). Dogs with lower expression of MFI of CD11b inside CD14 monocytes were represented by dogs with parasite ear tissue load and able to infect phlebotomines (IHQ⁺/XENO⁺) (p = 0,0032). Comparable results were obtained for MFI of MHCII (p = 0.0054). In addition, considering the population frequency of CD11b⁺TLR2⁺ and CD11b⁺MHCII⁺, higher values were obtained from dogs with IHQ⁻/XENO⁻ than dogs with IHQ⁺/XENO⁺ (p = 0.01; p = 0.0048, respectively). These data, together with the TLR2 and NO assays results (CD11b⁺TLR2⁺ and NO with higher values for dogs with IHQ⁻/XENO⁻ than dogs with IHQ⁺/XENO⁺, led to the conclusion that IHQ⁻/XENO⁻ dogs are more resistant or could modulate the cellular immune response essential for Leishmania tissue clearance.