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Despite the intramuscular route being the most used vaccination strategy against SARS-CoV-2, the intradermal route has been studied around the globe as a strong candidate for immunization against SARS-CoV-2. Adjuvants have shown to be essential vaccine components that are capable of driving robust immune responses and increasing the vaccination efficacy. In this work, our group aimed to develop a vaccination strategy for SARS-CoV-2 using a trimeric spike protein, by testing the best route with formulations containing the adjuvants AddaS03, CpG, MPL, Alum, or a combination of two of them. Our results showed that formulations that were made with AddaS03 or CpG alone or AddaS03 combined with CpG were able to induce high levels of IgG, IgG1, and IgG2a; high titers of neutralizing antibodies against SARS-CoV-2 original strain; and also induced high hypersensitivity during the challenge with Spike protein and a high level of IFN-γ producing CD4 + T-cells in mice. Altogether, those data indicate that AddaS03, CpG, or both combined may be used as adjuvants in vaccines for COVID-19.

The SARS-CoV-2 pandemic has had a social and economic impact worldwide, and vaccination is an efficient strategy for diminishing those damages. New adjuvant formulations are required for the high vaccine demands, especially adjuvant formulations that induce a Th1 phenotype. Herein we assess a vaccination strategy using a combination of Alum and polyinosinic:polycytidylic acid [Poly(I:C)] adjuvants plus the SARS-CoV-2 spike protein in a prefusion trimeric conformation by an intradermal (ID) route. We found high levels of IgG anti-spike antibodies in the serum by enzyme linked immunosorbent assay (ELISA) and high neutralizing titers against SARS-CoV-2 in vitro by neutralization assay, after two or three immunizations. By evaluating the production of IgG subtypes, as expected, we found that formulations containing Poly(I:C) induced IgG2a whereas Alum did not. The combination of these two adjuvants induced high levels of both IgG1 and IgG2a. In addition, cellular immune responses of CD4 + and CD8 + T cells producing interferon-gamma were equivalent, demonstrating that the Alum + Poly(I:C) combination supported a Th1 profile. Based on the high neutralizing titers, we evaluated B cells in the germinal centers, which are specific for receptor-binding domain (RBD) and spike, and observed that more positive B cells were induced upon the Alum + Poly(I:C) combination. Moreover, these B cells produced antibodies against both RBD and non-RBD sites. We also studied the impact of this vaccination preparation [spike protein with Alum + Poly(I:C)] in the lungs of mice challenged with inactivated SARS-CoV-2 virus. We found a production of IgG, but not IgA, and a reduction in neutrophil recruitment in the bronchoalveolar lavage fluid (BALF) of mice, suggesting that our immunization scheme reduced lung inflammation. Altogether, our data suggest that Alum and Poly(I:C) together is a possible adjuvant combination for vaccines against SARS-CoV-2 by the intradermal route., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 dos-Santos, Firmino-Cruz, da Fonseca-Martins, Oliveira-Maciel, Perez, Roncaglia-Pereira, Dumard, Guedes-da-Silva, Santos, Leandro, Ferreira, Guimarães-Pinto, Conde, Rodrigues, Silva, Alvim, Lima, Marsili, Abreu, Ferreira Jr., Mohana Borges, Tanuri, Souza, Rossi-Bergmann, Vale, Silva, de Oliveira, Filardy, Gomes and de Matos Guedes.)

We used the recombinant trimeric spike (S) glycoprotein in the prefusion conformation to immunize horses for the production of hyperimmune globulins against SARS-CoV-2. Serum antibody titers measured by ELISA were above 1:10 6 , and the neutralizing antibody titer against authentic virus (WT) was 1:14,604 (average PRNT 90 ). Plasma from immunized animals was pepsin digested to remove the Fc portion and purified, yielding an F(ab') 2 preparation with PRNT 90 titers 150-fold higher than the neutralizing titers in human convalescent plasma. Challenge studies were carried out in hamsters and showed the in vivo ability of equine F(ab') 2 to reduce viral load in the pulmonary tissues and significant clinical improvement determined by weight gain. The neutralization curve by F(ab') 2 was similar against the WT and P.2 variants, but displaced to higher concentrations by 0.39 log units against the P.1 (Gamma) variant. These results support the possibility of using equine F(ab') 2 preparation for the clinical treatment of COVID patients., Competing Interests: The authors declare no competing interest., (© 2021 The Author(s).)

Zika virus (ZIKV) emerged as an important infectious disease agent in Brazil in 2016. Infection usually leads to mild symptoms, but severe congenital neurological disorders and Guillain-Barré syndrome have been reported following ZIKV exposure. Creating an effective vaccine against ZIKV is a public health priority. We describe the protective effect of an already licensed attenuated yellow fever vaccine (YFV, 17DD) in type-I interferon receptor knockout mice (A129) and immunocompetent BALB/c and SV-129 (A129 background) mice infected with ZIKV. YFV vaccination provided protection against ZIKV, with decreased mortality in A129 mice, a reduction in the cerebral viral load in all mice, and weight loss prevention in BALB/c mice. The A129 mice that were challenged two and three weeks after the first dose of the vaccine were fully protected, whereas partial protection was observed five weeks after vaccination. In all cases, the YFV vaccine provoked a substantial decrease in the cerebral viral load. YFV immunization also prevented hippocampal synapse loss and microgliosis in ZIKV-infected mice. Our vaccine model is T cell-dependent, with AG129 mice being unable to tolerate immunization (vaccination is lethal in this mouse model), indicating the importance of IFN-γ in immunogenicity. To confirm the role of T cells, we immunized nude mice that we demonstrated to be very susceptible to infection. Immunization with YFV and challenge 7 days after booster did not protect nude mice in terms of weight loss and showed partial protection in the survival curve. When we evaluated the humoral response, the vaccine elicited significant antibody titers against ZIKV; however, it showed no neutralizing activity in vitro and in vivo. The data indicate that a cell-mediated response promotes protection against cerebral infection, which is crucial to vaccine protection, and it appears to not necessarily require a humoral response. This protective effect can also be attributed to innate factors, but more studies are needed to strengthen this hypothesis. Our findings open the way to using an available and inexpensive vaccine for large-scale immunization in the event of a ZIKV outbreak., Competing Interests: The authors have declared that no competing interests exist.

Lactoferrin is part of the innate immune system, with antiviral activity against numerous DNA and RNA viruses. Rhinoviruses, the leading cause of the common cold, are associated with exacerbation of respiratory illnesses such as asthma. Here, we explored the effect of bovine lactoferrin (BLf) on RV-B14 infectivity. Using different assays, we show that the effect of BLf is strongest during adhesion of the virus to the cell and entry. Tracking the internalisation of BLf and virus revealed a degree of colocalisation, although their interaction was only confirmed in vitro using empty viral particles, indicating a possible additional influence of BLf on other infection steps.

Enveloped viruses rely on different lipid classes present in cell membranes to accomplish several steps of their life cycle in the host. Particularly for alphaviruses, a medically important group of arboviruses, which are part of the Togaviridae family, cholesterol seems to be a critical lipid exploited during infection, although its relevance may vary depending on which stage of the virus life cycle is under consideration and whether infection takes place in vertebrate or invertebrate hosts. In this review, the role of cholesterol in both early and late events of alphavirus infection and how viral replication may affect cholesterol metabolism are summarized, taking into account studies on Old World and New World alphaviruses in different cell lines. Moreover, the importance of cholesterol for the structural stability of alphavirus particles is also discussed, shedding light on the role played by this lipid when they leave the host cell.

Recognition and internalisation of intracellular pathogens by host cells is a multifactorial process, involving both stable and transient interactions. The plasticity of the host cell plasma membrane is fundamental in this infectious process. Here, the participation of macrophage lipid microdomains during adhesion and internalisation of the fungal pathogen Histoplasma capsulatum (Hc) was investigated. An increase in membrane lateral organisation, which is a characteristic of lipid microdomains, was observed during the first steps of Hc-macrophage interaction. Cholesterol enrichment in macrophage membranes around Hc contact regions and reduced levels of Hc-macrophage association after cholesterol removal also suggested the participation of lipid microdomains during Hc-macrophage interaction. Using optical tweezers to study cell-to-cell interactions, we showed that cholesterol depletion increased the time required for Hc adhesion. Additionally, fungal internalisation was significantly reduced under these conditions. Moreover, macrophages treated with the ceramide-glucosyltransferase inhibitor (P4r) and macrophages with altered ganglioside synthesis (from B4galnt1 -/- mice) showed a deficient ability to interact with Hc. Coincubation of oligo-GM1 and treatment with Cholera toxin Subunit B, which recognises the ganglioside GM1, also reduced Hc association. Although purified GM1 did not alter Hc binding, treatment with P4 significantly increased the time required for Hc binding to macrophages. The content of CD18 was displaced from lipid microdomains in B4galnt1 -/- macrophages. In addition, macrophages with reduced CD18 expression (CD18 low ) were associated with Hc at levels similar to wild-type cells. Finally, CD11b and CD18 colocalised with GM1 during Hc-macrophage interaction. Our results indicate that lipid rafts and particularly complex gangliosides that reside in lipid rafts stabilise Hc-macrophage adhesion and mediate efficient internalisation during histoplasmosis., (© 2018 John Wiley & Sons Ltd.)

The functionality of the tumor suppressor p53 is altered in more than 50% of human cancers, and many individuals with cancer exhibit amyloid-like buildups of aggregated p53. An understanding of what triggers the pathogenic amyloid conversion of p53 is required for the further development of cancer therapies. Here, perturbation of the p53 core domain (p53C) with subdenaturing concentrations of guanidine hydrochloride and high hydrostatic pressure revealed native-like molten globule (MG) states, a subset of which were highly prone to amyloidogenic aggregation. We found that MG conformers of p53C, probably representing population-weighted averages of multiple states, have different volumetric properties, as determined by pressure perturbation and size-exclusion chromatography. We also found that they bind the fluorescent dye 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and have a native-like tertiary structure that occludes the single Trp residue in p53. Fluorescence experiments revealed conformational changes of the single Trp and Tyr residues before p53 unfolding and the presence of MG conformers, some of which were highly prone to aggregation. p53C exhibited marginal unfolding cooperativity, which could be modulated from unfolding to aggregation pathways with chemical or physical forces. We conclude that trapping amyloid precursor states in solution is a promising approach for understanding p53 aggregation in cancer. Our findings support the use of single-Trp fluorescence as a probe for evaluating p53 stability, effects of mutations, and the efficacy of therapeutics designed to stabilize p53., (© 2018 Pedrote et al.)

The global situation of diseases transmitted by arthropod-borne viruses such as Dengue (DENV), Yellow Fever (YFV), Chikungunya (CHIKV) and Zika (ZIKV) viruses is alarming and treatment of human infection by these arboviruses faces several challenges. The discovery of broad-spectrum antiviral molecules, able to inactivate different groups of viruses, is an interesting approach. The viral envelope is a common structure among arboviruses, being a potential target for antivirals. Porphyrins are amphipathic molecules able to interact with membranes and absorb light, being widely used in photodynamic therapy. Previously, we showed that heme, Co-protoporphyrin IX (CoPPIX) and Sn-protoporphyrin IX (SnPPIX) directly inactivate DENV and YFV infectious particles. Here we demonstrate that the antiviral activity of these porphyrins can be broadened to CHIKV, ZIKV, Mayaro virus, Sindbis virus and Vesicular Stomatitis virus. Porphyrin treatment causes viral envelope protein loss, affecting viral morphology, adsorption and entry into target cells. Also, light-stimulation enhanced the SnPPIX activity against all tested arboviruses. In summary, CoPPIX and SnPPIX were shown to be efficient broad-spectrum compounds to inactivate medically and veterinary important viruses.

Background: Avian influenza A viruses can cross naturally into mammals and cause severe diseases, as observed for H5N1. The high lethality of human infections causes major concerns about the real risk of a possible pandemic of severe diseases to which human susceptibility may be high and universal. High hydrostatic pressure (HHP) is a valuable tool for studies regarding the folding of proteins and the assembly of macromolecular structures such as viruses; furthermore, HHP has already been demonstrated to promote viral inactivation., Methods: Here, we investigated the structural stability of avian and human influenza viruses using spectroscopic and light-scattering techniques. We found that both particles have similar structural stabilities and that HHP promotes structural changes., Results: HHP induced slight structural changes to both human and avian influenza viruses, and these changes were largely reversible when the pressure returned to its initial level. The spectroscopic data showed that H3N2 was more pressure-sensitive than H3N8. Structural changes did not predict changes in protein function, as H3N2 fusion activity was not affected, while H3N8 fusion activity drastically decreased. The fusion activity of H1N1 was also strongly affected by HHP. In all cases, HHP caused inactivation of the different influenza viruses., Conclusions: HHP may be a useful tool for vaccine development, as it induces minor and reversible structural changes that may be associated with partial preservation of viral biological activities and may potentiate their immunogenic response while abolishing their infectivity. We also confirmed that, although pressure does not promote drastic changes in viral particle structure, it can distinctly affect viral fusion activity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Alphaviruses are enveloped arboviruses mainly proposed to infect host cells by receptor-mediated endocytosis followed by fusion between the viral envelope and the endosomal membrane. The fusion reaction is triggered by low pH and requires the presence of both cholesterol and sphingolipids in the target membrane, suggesting the involvement of lipid rafts in the cell entry mechanism. In this study, we show for the first time the interaction of an enveloped virus with membrane microdomains isolated from living cells. Using Mayaro virus (MAYV), a New World alphavirus, we verified that virus fusion to these domains occurred to a significant extent upon acidification, although its kinetics was quite slow when compared to that of fusion with artificial liposomes demonstrated in a previous work. Surprisingly, when virus was previously exposed to acidic pH, a condition previously shown to inhibit alphavirus binding and fusion to target membranes as well as infectivity, and then reneutralized, its ability to fuse with membrane microdomains at low pH was retained. Interestingly, this observation correlated with a partial reversion of low pH-induced conformational changes in viral proteins and retention of virus infectivity upon reneutralization. Our results suggest that MAYV entry into host cells could alternatively involve internalization via lipid rafts and that the conformational changes triggered by low pH in the viral spike proteins during the entry process are partially reversible.

Chikungunya (CHIKV) and Zika (ZIKV) viruses are arboviruses which have recently broken their sylvatic isolation and gone on to spread rampantly among humans in some urban areas of the world, especially in Latin America. Given the lack of effective interventions against such viruses, the aim of this work was to evaluate the antiviral potential of bovine lactoferrin (bLf) in their infections. Through viability, plaque, immunofluorescence and nucleic acid quantification assays, our data show that bLf exerts a dose-dependent strong inhibitory effect on the infection of Vero cells by the aforementioned arboviruses, reducing their infection efficiency by up to nearly 80 %, with no expressive cytotoxicity, and that such antiviral activity occurs at the levels of input and output of virus particles. These findings reveal that bLf antimicrobial properties are extendable to CHIKV and ZIKV, underlining a generic inhibition mechanism that can be explored to develop a potential strategy against their infections.

Virus resistance to antiviral therapies is an increasing concern that makes the development of broad-spectrum antiviral drugs urgent. Targeting of the viral envelope, a component shared by a large number of viruses, emerges as a promising strategy to overcome this problem. Natural and synthetic porphyrins are good candidates for antiviral development due to their relative hydrophobicity and pro-oxidant character. In the present work, we characterized the antiviral activities of protoprophyrin IX (PPIX), Zn-protoporphyrin IX (ZnPPIX), and mesoporphyrin IX (MPIX) against vesicular stomatitis virus (VSV) and evaluated the mechanisms involved in this activity. Treatment of VSV with PPIX, ZnPPIX, and MPIX promoted dose-dependent virus inactivation, which was potentiated by porphyrin photoactivation. All three porphyrins inserted into lipid vesicles and disturbed the viral membrane organization. In addition, the porphyrins also affected viral proteins, inducing VSV glycoprotein cross-linking, which was enhanced by porphyrin photoactivation. Virus incubation with sodium azide and α-tocopherol partially protected VSV from inactivation by porphyrins, suggesting that singlet oxygen ( 1 O 2 ) was the main reactive oxygen species produced by photoactivation of these molecules. Furthermore, 1 O 2 was detected by 9,10-dimethylanthracene oxidation in photoactivated porphyrin samples, reinforcing this hypothesis. These results reveal the potential therapeutic application of PPIX, ZnPPIX, and MPIX as good models for broad antiviral drug design., (Copyright © 2017 American Society for Microbiology.)

Mayaro virus (MAYV) is an emergent sylvatic alphavirus in South America, related to sporadic outbreaks of a chikungunya-like human febrile illness accompanied by severe arthralgia. Despite its high potential for urban emergence, MAYV is still an obscure virus with scarce information about its infection cycle, including the corresponding early events. Even for prototypical alphaviruses, the cell entry mechanism still has some rough edges to trim: although clathrin-mediated endocytosis is quoted as the putative route, alternative paths as distinct as direct virus genome injection through the cell plasma membrane seems to be possible. Our aim was to clarify crucial details on the entry route exploited by MAYV to gain access into the host cell. Tracking the virus since its first contact with the surface of Vero cells by fluorescence microscopy, we show that its entry occurs by a fast endocytic process and relies on fusion with acidic endosomal compartments. Moreover, blocking clathrin-mediated endocytosis or depleting cholesterol from the cell membrane leads to a strong inhibition of viral infection, as assessed by plaque assays. Following this clue, we found that early endosomes and caveolae-derived vesicles are both implicated as target membranes for MAYV fusion. Our findings unravel the very first events that culminate in a productive infection by MAYV and shed light on potential targets for a rational antiviral therapy, besides providing a better comprehension of the entry routes exploited by alphaviruses to get into the cell., Competing Interests: Jerson L. Silva is an Academic Editor for PeerJ.

Background: Hepatitis C virus (HCV) core protein, in addition to its structural role to form the nucleocapsid assembly, plays a critical role in HCV pathogenesis by interfering in several cellular processes, including microRNA and mRNA homeostasis. The C-terminal truncated HCV core protein (C124) is intrinsically unstructured in solution and is able to interact with unspecific nucleic acids, in the micromolar range, and to assemble into nucleocapsid-like particles (NLPs) in vitro . The specificity and propensity of C124 to the assembly and its implications on HCV pathogenesis are not well understood., Methods: Spectroscopic techniques, transmission electron microscopy and calorimetry were used to better understand the propensity of C124 to fold or to multimerize into NLPs when subjected to different conditions or in the presence of unspecific nucleic acids of equivalent size to cellular microRNAs., Results: The structural analysis indicated that C124 has low propensity to self-folding. On the other hand, for the first time, we show that C124, in the absence of nucleic acids, multimerizes into empty NLPs when subjected to a pH close to its isoelectric point (pH ≈ 12), indicating that assembly is mainly driven by charge neutralization. Isothermal calorimetry data showed that the assembly of NLPs promoted by nucleic acids is enthalpy driven. Additionally, data obtained from fluorescence correlation spectroscopy show that C124, in nanomolar range, was able to interact and to sequester a large number of short unspecific nucleic acids into NLPs., Discussion: Together, our data showed that the charge neutralization is the major factor for the nucleocapsid-like particles assembly from C-terminal truncated HCV core protein. This finding suggests that HCV core protein may physically interact with unspecific cellular polyanions, which may correspond to microRNAs and mRNAs in a host cell infected by HCV, triggering their confinement into infectious particles., Competing Interests: Jerson Lima Silva is an Academic Editor for PeerJ.