A promiscuous T cell epitope-based HIV vaccine providing redundant population coverage of the HLA class II elicits broad, polyfunctional T cell responses in nonhuman primates.

Over the last few decades, several emerging or reemerging viral diseases with no readily available vaccines have ravaged the world. A platform to fastly generate vaccines inducing potent and durable neutralizing antibody and T cell responses is sorely needed. Bioinformatically identified epitope-based vaccines can focus on immunodominant T cell epitopes and induce more potent immune responses than a whole antigen vaccine and may be deployed more rapidly and less costly than whole-gene vaccines. Increasing evidence has shown the importance of the CD4+ T cell response in protection against HIV and other viral infections. The previously described DNA vaccine HIVBr18 encodes 18 conserved, promiscuous epitopes binding to multiple HLA-DR-binding HIV epitopes amply recognized by HIV-1-infected patients. HIVBr18 elicited broad, polyfunctional, and durable CD4+and CD8+ T cell responses in BALB/c and mice transgenic to HLA class II alleles, showing cross-species promiscuity. To fully delineate the promiscuity of the HLA class II vaccine epitopes, we assessed their binding to 34 human class II (HLA-DR, DQ, and -DP) molecules, and immunized nonhuman primates. Results ascertained redundant 100% coverage of the human population for multiple peptides. We then immunized Rhesus macaques with HIVBr18 under in vivo electroporation. The immunization induced strong, predominantly polyfunctional CD4+ T cell responses in all animals to 13 out of the 18 epitopes; T cells from each animal recognized 7-11 epitopes. Our results provide a preliminary proof of concept that immunization with a vaccine encoding epitopes with high and redundant coverage of the human population can elicit potent T cell responses to multiple epitopes, across species and MHC barriers. This approach may facilitate the rapid deployment of immunogens eliciting cellular immunity against emerging infectious diseases, such as COVID-19.

StreptInCor, a Group A Streptococcal Adsorbed Vaccine: Evaluation of Repeated Intramuscular Dose Toxicity Testing in Rats.

Streptococcus pyogenes infections continue to be a worldwide public health problem, causing various diseases in humans, with rheumatic fever and rheumatic heart disease being the most harmful manifestations. Impetigo and post-streptococcal glomerulonephritis are also important sequelae of skin infections. We have developed a candidate vaccine epitope (StreptInCor) that presents promising results in diverse animal models. To assess whether the StreptInCor alum-adsorbed vaccine could induce undesirable effects, a certified independent company conducted a repeated intramuscular dose toxicity evaluation in Wistar rats, a choice model for toxicity studies. We did not observe significant alterations in clinical, hematological, biochemical, anatomical, or histopathological parameters due to vaccine administration, even when the animals received the highest dose. In conclusion, repeated intramuscular doses did not show signs of macroscopic or other significant changes in the clinical or histopathological parameters, indicating that StreptInCor can be considered a safe candidate vaccine.

Behavioral effects of LPS in adult, middle-aged and aged mice.

Acute infections lead to alterations in behavior, collectively known as sickness behavior, which includes reduction in locomotion, food ingestion, sexual and social behavior, environmental exploration, and sleep profile. Although generally seen as undesired, sickness behavior represents a conserved strategy for animals to overcome disease. Aging process is associated with a variety of changes in immunity, which are referred to as immunosenescence, and include higher mortality by infectious diseases. Few works studied sickness behavior display in old animals. Thus, we sought to investigate the display of sickness related behaviors on aged mice. Adult (3-6 months old), middle-aged (12-15 m) and aged mice (18-22 m) were treated with i.p. LPS (200 microg/kg) and their behaviors were assessed in the open field and in the elevated plus-maze. Exploratory activity was similar in aged mice treated or not with LPS in both apparati. In the open field, locomotion remained at baseline levels; in the elevated plus-maze, there was a time-dependent decrease in motor activity.

Protein-energy malnutrition decreases the expression of TLR-4/MD-2 and CD14 receptors in peritoneal macrophages and reduces the synthesis of TNF-alpha in response to lipopolysaccharide (LPS) in mice.

Protein-energy malnutrition (PEM) modifies resistance to infection, impairing a number of physiological processes, including hematopoiesis. In this study, we examined a few aspects of the inflammatory response to LPS in a model of PEM. We evaluated the cellularity of the blood, bone marrow and spleen, as well as phagocytic, fungicidal and spreading activity, the production in vivo and in vitro of TNF-alpha, IL-1alpha and IL-6, and the expression of CD14 and TLR-4/MD-2 receptors in macrophages. Two-month-old male Swiss mice were submitted to PEM with a low-protein diet containing 4% protein as compared to 20% protein in the control diet. When the experimental group had attained about 20% loss of their original body weight, they were used in the experiments. Malnourished animals presented anemia, leucopenia and severe reduction in bone marrow, spleen and peritoneal cavity cellularity. The production of TNF-alpha, IL-1alpha and IL-6 stimulated in vivo with LPS and the production of IL-6 in bone marrow cells cultured with LPS and the production of TNF-alpha in bone marrow, spleen and peritoneal cells cultured with LPS were significantly lower in malnourished animals. The expression of CD14 and TLR-4/MD-2 receptors was found to be significantly lower in macrophages of malnourished animals. These findings suggest that malnourished animals present a deficient response to LPS. The lower expression of the CD14 and TLR-4/MD-2 receptors may be partly responsible for the immunodeficiency observed in the malnourished mice. These data lead us to infer that the nutritional state interferes with the activation of macrophages and with the capacity to mount an immune response.

Neural correlates of IgE-mediated allergy.

Although many authors have considered a direct interaction between allergic reactions and behavioral changes, supporting evidence has been elusive. In this series of studies we show that after oral or nasal ovalbumin (OVA) challenge, allergic mice present increased Fos expression in the paraventricular nucleus of the hypothalamus (PVN) and in the central nucleus of the amygdala (CeA). Mice with food allergy display higher levels of anxiety and increased serum corticosterone levels, and allergy-activated neurons express corticotropin-releasing factor (CRF) in the PVN and CeA. OVA-allergic mice develop aversion to an antigen-containing solution, and also avoid a dark compartment previously associated with nebulized OVA. Results on brain Fos expression and behavioral data seem compatible with adaptive responses. Removal of IgE by either antibody depletion or the development of oral tolerance precluded all responses analyzed here. C-sensitive fiber destruction by neonatal capsaicin inhibited the activation in the PVN, but not in the CeA, and decreased the magnitude of food aversion. Cromolyn, a mast cell stabilizer, completely blocked Fos expression in the PVN and CeA, and precluded the development of aversion to the dark compartment associated with nebulized OVA. Employing mice that do not develop an important inflammatory infiltrate following nasal OVA challenge, we found that inflammatory cells are not required at the site of challenge in order to trigger neural or behavioral correlates of murine experimental asthma. Altogether, we have built a solid foundation for understanding neuroimmune interactions during allergic responses that may contribute to the comprehension of psychological disorders associated with allergy.

Differential effects of lipopolysaccharide in the social behavior of dominant and submissive mice.

Acutely infected animals show a set of non-specific behavioral changes known as sickness behavior. Recent studies have shown that occurrence of sickness behavior is regulated according to a motivational perspective. Thus, the display of sickness behavior may compete with display of other behaviors. In this work, we sought to determine the effects of lipopolysaccharide (LPS) administration (15 microg/mouse i.p.) in the social behavior of dominant and submissive mice. Results showed that social hierarchy influences the expression of sickness behavior. While dominant mice treated with LPS showed an expected reduction in total frequency of behaviors displayed, such decrease did not happen following the same treatment to submissive mice. Similar results occurred regarding social and aggressive behavior. The use of a motivational perspective provides the assumption that, due to their high social ranking, dominant mice were able to prioritize recuperative behavior. Submissive mice, on the other hand, even though treated with LPS, seemed to essentially focus on social defensive behaviors since they remained in the presence of the dominant individuals. Effects of sickness on the hierarchical organization of mice remain to be further investigated.

Neural correlates of IgE-mediated food allergy.

Although many authors have considered the possibility of a direct interaction between food allergy and behavioral changes, the evidence supporting this hypothesis is elusive. Here, we show that after oral ovalbumin (OVA) challenge, allergic mice present higher levels of anxiety, increased Fos expression in emotionality-related brain areas, and aversion to OVA-containing solution. Moreover, treatment with anti-IgE antibody or induction of oral tolerance abrogate both food aversion and the expression of c-fos in the central nervous system (CNS). Our findings establish a direct relationship between brain function and food allergy, thus creating a solid ground for understanding the etiology of psychological disorders in allergic patients.

Neural pathways involved in food allergy signaling in the mouse brain: role of capsaicin-sensitive afferents.

There is increasing evidence supporting the notion that brain-gut communication is crucial for the manifestation of functional gastrointestinal (GI) disorders. Employing denervation by neonatal capsaicin treatment, we investigated here the role of unmyelinated C-fibers in food allergy signaling in the brain. We found that 90 min after oral ovalbumin (OVA) challenge, allergic mice present increased c-fos expression in emotionality-related brain areas such as the paraventricular nucleus of the hypothalamus (PVN) and the central nucleus of the amygdala (CeA). Food allergy also induced enhanced Fos immunoreactivity in the nucleus of tractus solitarii (NTS) of OVA-immunized animals. We also show that while the degree of Fos staining in the NTS of allergic mice was only diminished by neonatal capsaicin, it was completely blocked in the PVN. However, capsaicin did not modify food allergy-induced c-fos expression in the CeA. In conclusion, this study provides evidence showing that unmyelinated C-fibers are part of the neural pathways involved in food allergy-induced activation of specific brain areas, particularly the PVN and to a lesser extent the NTS.

Effects of acute and long-term diazepam administrations on neutrophil activity: a flow cytometric study.

This study analyzed the effects of acute and long-term diazepam treatments on rat peripheral blood neutrophil activity and cortisol serum levels. Rats were acutely and long-term (21 days, once daily) treated with diazepam (10 mg/kg) or its vehicle (1.0 ml/kg). Blood was collected 1 h after treatments for flow cytometric analysis of neutrophil oxidative burst and phagocytosis. Corticosterone and diazepam concentrations were also determined. Results showed that: (1) both diazepam treatments increased lipopolysaccharide (LPS) and phorbol myristate acetate (PMA)-induced neutrophil oxidative burst; (2) the increase in oxidative burst after Staphylococcus aureus induction in acutely treated animals was higher than that observed after long-term treatment; (3) phagocytosis is increased by acute diazepam treatment and decreased by a long-term regimen; (4) acute, but not long-term, diazepam treatment increased corticosterone levels; (5) diazepam plasmatic levels after acute and long-term treatments were not different. These results indicate the development of tolerance to diazepam effects on corticosterone serum levels but not on neutrophil activity.

Lipopolysaccharide administration in the dominant mouse destabilizes social hierarchy.

Sickness behavior is a set of behavioral changes that are part of an adaptive strategy to overcome infection. Mice that interact with conspecifics displaying sickness behavior also show relevant behavioral changes. In this work we sought to determine the role of sickness behavior display by a dominant mouse as a promoter of hierarchy instability. We treated the dominant mouse within a dyad with lipopolysaccharide (LPS) (400 µg/kg, i.p.) for three consecutive days and assessed social dominance behavior. Since elder animals display increased inflammatory responses and the behaviors toward conspecifics are influenced by kinship we also assessed whether kinship and age, might influence sickness related hierarchy instability. Our results show that administration of LPS in the dominant mouse promotes social instability within a dyad, and indicates that this instability could be influenced by kinship and age.

Behavior: a relevant tool for brain-immune system interaction studies.

Neuroimmunomodulation describes the field focused on understanding the mechanisms by which the central nervous system interacts with the immune system, potentially leading to changes in animal behavior. Nonetheless, not many articles dealing with neuroimmunomodulation employ behavior as an analytical endpoint. Even fewer papers deal with social status as a possible modifier of neuroimmune phenomena. In the described sets of experiments, we tackle both, using a paradigm of social dominance and subordination. We first review data on the effects of different ranks within a stable hierarchical relationship. Submissive mice in this condition display more anxiety-like behaviors, have decreased innate immunity, and show a decreased resistance to implantation and development of melanoma metastases in their lungs. This suggests that even in a stable, social, hierarchical rank, submissive animals may be subjected to higher levels of stress, with putative biological relevance to host susceptibility to disease. Second, we review data on how dominant and submissive mice respond differentially to lipopolysaccharide (LPS), employing a motivational perspective to sickness behavior. Dominant animals display decreased number and frequency in several aspects of behavior, particularly agonistic social interaction, that is, directed toward the submissive cage mate. This was not observed in submissive mice that maintained the required behavior expected by its dominant mate. Expression of sickness behavior relies on motivational reorganization of priorities, which are different along different social ranks, leading to diverse outcomes. We suggest that in vitro assessment of neuroimmune phenomena can only be understood based on the behavioral context in which they occur.

Cohabitation with a sick cage mate: consequences on behavior and on ehrlich tumor growth.

The present study analyzed the effects of cohabitation for 11 days with a sick cage mate on behavior and Ehrlich tumor growth in mice. Pairs of female mice were divided into one control and one experimental group. One mouse of each control pair was kept undisturbed and called 'healthy companion' (HC). One animal of each experimental pair of mice was inoculated (i.p.) with 5 x 10(6) Ehrlich tumor cells, and the other, the object of this study, was called 'sick companion' (SC). The SC mice presented: (1) increased activity in an open field, (2) increased number of entries and of movements within the plus-maze open arms, (3) similar levels of plus-maze closed-arm exploration, (4) a decrease in the exploratory activity in a hole board, (5) a decrease in the number of white but not red blood cells, and (6) similar corticosterone serum levels. Eleven days after cohabitation with a conspecific, HC and SC mice were injected with 5 x10(6) Ehrlich tumor cells. Results showed that SC animals presented decreased resistance to the ascitic form of the Ehrlich tumor. The observed data provide experimental evidence that psychosocial stress induced by cohabitation with a sick cage mate changed at the same time some behavioral and physiological parameters, and decreased resistance to Ehrlich tumor. These data are discussed in the light of a possible neuroimmune system interaction.

Influência da leucose enzoótica bovina na função fagocítica de leucócitos circulantes em animais manifestando linfocitose persistente / Influence of enzootic bovine leukosis on phagocytic function of circulating leukocytes from animals with persistent lymphocytosis

Avaliou-se, por citometria de fluxo, a fagocitose de Staphylococcus aureus conjugados com iodeto de propídio (IP), por leucócitos circulantes obtidos de cinco fêmeas bovinas negativas no sorodiagnóstico para a Leucose Enzoótica Bovina (LEB); de cinco fêmeas infectadas, manifestando linfocitose persistente (LP); e de cinco fêmeas infectadas, porém alinfocitóticas. Observou-se que, entre as amostras dos animais soronegativos, a porcentagem média de células realizando fagocitose (12,90%) não diferiu da observada entre as células dos animais alinfocitóticos (14,70%). Contudo, ambas foram maiores (p=0,047) que aquela verificada entre as células obtidas de animais manifestando LP (7,20%). Além disso, a intensidade média de fagocitose (caracterizada pela intensidade de fluorescência do IP, em valores arbitrários), verificada em leucócitos de animais manifestando LP (17,43) foi menor (p<0,001) que a observada em leucócitos de animais alinfocitóticos (29,50), e que a observada em leucócitos de animais soronegativos (25,18), que não diferiram entre si. Assim, os resultados permitem-nos alvitrar que há alteração na função fagocítica de leucócitos circulantes em animais infectados pelo vírus da LEB, manifestando LP.

This study evaluated the phagocytosis of propidium iodide-labeled Staphylococcus aureus (PI-Sa) by circulating leukocytes obtained from five Enzootic Bovine Leukosis (EBL)-negative cows, five naturally EBL-infected, non-lymphocytotic cows, and five EBL-positive cows with persistent lymphocytosis (PL), analyzed by flow cytometry. Among cells obtained from EBL-infected cows, presenting PL, the percentage of leukocytes carrying out phagocytosis (7.20%), was smaller (p=0.047) than that verified among cells obtained from non-infected (12.90%), and from BLV-infected, non-lymphocytotic cows (14.70%). Furthermore, leukocytes obtained from EBL-infected cows, presenting PL, showed smaller phagocytosis intensity (characterized by the intensity of propidium iodide fluorescence) than leukocytes obtained from non-infected and from EBL-infected, non-lymphocytotic, cows (p<0.001). Therefore, results show a decreased phagocytic function among circulating leukocytes obtained from BLV-infected, lymphocytotic cows.