Pharmacological inhibition of UPR sensor PERK attenuates HIV Tat-induced inflammatory M1 phenotype in microglial cells.

HIV-1-associated neurocognitive disorders (HAND) are a major concern in HIV-infected individuals despite the currently available antiretroviral therapy regime. Impaired M1 pro-inflammatory microglial activation is considered one of the hallmark features of HAND neuropathogenesis, and it has been suggested that circulant HIV-1 transactivator protein (Tat) can play a critical role in this process. At the same time, endoplasmatic reticulum (ER) stress has also been implicated in neurodegenerative conditions resulting from the accumulation of misfolded proteins and subsequent unfolded protein response (UPR) deflagration. Here, we demonstrate that pharmacological inhibition of UPR-related protein kinase R-like endoplasmic reticulum kinase (PERK) can attenuate HIV-1 Tat-induced M1 inflammatory state in microglia in vitro. Our initial experiments demonstrate that the bystander stimulus of recombinant Tat on BV-2 microglial cells result in the coupled overexpression of central UPR markers and pro-inflammatory mediators such as iNOS, surface CD16/32 and secreted tumour necrosis factor-α (TNF-α), IL-6, monocyte chemoattractant protein (MCP)-1 and NO. We show that blocking PERK-eIF2-α-ATF4 signalling using the PERK inhibitor GSK2606414 leads to reduced inflammatory response in M1-like BV-2 cells activated by recombinant Tat. Taken together, these findings suggest that PERK targeting may provide a therapeutic intervention to mitigate against lasting neuroinflammation and neuronal loss in of HAND.

A Type IIa crustin from the pink shrimp Farfantepenaeus paulensis (crusFpau) is constitutively synthesized and stored by specific granule-containing hemocyte subpopulations.

Crustins are cysteine-rich antimicrobial peptides (AMPs) widely distributed across crustaceans. From the four described crustin Types (I to IV), crustins from the subtype IIa are the most abundant and diverse members found in penaeid shrimp. Despite the critical role of Type IIa crustins in shrimp antimicrobial defenses, there is still limited information about their synthesis and antimicrobial properties. Here, we report the subcellular localization and the antibacterial spectrum of crusFpau, a Type IIa crustin from the pink shrimp Farfantepenaeus paulensis. The recombinantly expressed crusFpau showed antimicrobial activity against both Gram-positive and Gram-negative bacteria at low concentrations. Results from immunofluorescence using anti-rcrusFpau antiserum revealed that crusFpau is synthetized and stored by both granular and semigranular hemocytes, but not by hyaline cells. Interestingly, not all granular and semigranular hemocytes stained for crusFpau, revealing that this crustin is produced by specific granule-containing hemocyte subpopulations. Finally, we showed that the granule-stored peptides are not constitutively secreted into the plasma of healthy animals.

HIV-1 Tat-induced bystander apoptosis in Jurkat cells involves unfolded protein responses.

The HIV transactivator protein (Tat) is a multifunctional protein that plays a critical role in viral replication and contributes to several pathological symptoms of HIV-1 infection, which has the loss of CD4+ T lymphocytes as one of its hallmark features. It has been shown that endoplasmic reticulum (ER) stress, including viral infections, is implicated in cellular dysfunction and cell death through activation of the unfolded protein response (UPR). Here, we demonstrate that the bystander stimulus of Tat on Jurkat cells resulted in time-dependent overexpression of major UPR markers including ER chaperone BiP, ER stress sensors ATF6, PERK, and IRE1, as well as an increase in levels of downstream mediators eIF2α, ATF4, XBP-1, and proapoptotic factors CHOP, GADD34, and BIM. This upregulation of UPR mediators was accompanied by decreased cell viability and increased apoptosis as evidenced by blue trypan dye exclusion and flow cytometry assays, respectively. Furthermore, we found that the Tat-associated apoptosis of Jurkat cells led to the loss of mitochondrial membrane potential and caspase-12 and -3 activation. Taken together, these results suggest that the exposure of HIV-1 Tat leads to ER stress/UPR triggering which in turn contributes to apoptotic death in Jurkat cells. SIGNIFICANCE OF THE STUDY: In the present work, we provide a substantial insight into the link between ER impairment and apoptotic death following a bystander HIV Tat stimulus, revealing an underlying ER-mediated apoptotic mechanism which could explain the continuous depletion of uninfected CD4+ T lymphocytes observed in HIV-related disease. Our findings reinforce the relevance of ER stress molecular responses in the course of HIV infection and may afford valuable information for the development of new therapeutic strategies to avoid CD4+ T lymphocyte loss and other disorders induced by circulant Tat.

Comprehensive Characterization of HIV-1 Molecular Epidemiology and Demographic History in the Brazilian Region Most Heavily Affected by AIDS.

UNLABELLED: The high incidence of AIDS cases and the dominance of HIV-1 subtype C infections are two features that distinguish the HIV-1 epidemic in the two southernmost Brazilian states (Rio Grande do Sul [RS] and Santa Catarina [SC]) from the epidemic in other parts of the country. Nevertheless, previous studies on HIV molecular epidemiology were conducted mainly in capital cities, and a more comprehensive understanding of factors driving this unique epidemic in Brazil is necessary. Blood samples were collected from individuals in 13 municipalities in the Brazilian southern region. HIV-1 env and pol genes were submitted to phylogenetic analyses for assignment of subtype, and viral population phylodynamics were reconstructed by applying Skygrid and logistic coalescent models in a Bayesian analysis. A high prevalence of subtype C was observed in all sampled locations; however, an increased frequency of recombinant strains was found in RS, with evidence for new circulating forms (CRFs). In the SC state, subtype B and C epidemics were associated with distinct exposure groups. Although logistic models estimated similar growth rates for HIV-1 subtype C (HIV-1C) and HIV-1B, a Skygrid plot reveals that the former epidemic has been expanding for a longer time. Our results highlight a consistent expansion of HIV-1C in south Brazil, and we also discuss how heterosexual and men who have sex with men (MSM) transmission chains might have impacted the current prevalence of HIV-1 subtypes in this region. IMPORTANCE: The AIDS epidemic in south Brazil is expanding rapidly, but the circumstances driving this condition are not well known. A high prevalence of HIV-1 subtype C was reported in the capital cities of this region, in contrast to the subtype B dominance in the rest of the country. This study sought to comparatively investigate the HIV-1 subtype B and C epidemics by sampling individuals from several cities in the two states with the highest AIDS incidences in Brazil. Our analyses showed distinct epidemic growth curves for the two epidemics, and we also found evidence suggesting that separate transmission chains may be impacting the viral phylodynamics and the emergence of new recombinant forms.

Contribution of Epidemiological Predictors in Unraveling the Phylogeographic History of HIV-1 Subtype C in Brazil.

UNLABELLED: The phylogeographic history of the Brazilian HIV-1 subtype C (HIV-1C) epidemic is still unclear. Previous studies have mainly focused on the capital cities of Brazilian federal states, and the fact that HIV-1C infections increase at a higher rate than subtype B infections in Brazil calls for a better understanding of the process of spatial spread. A comprehensive sequence data set sampled across 22 Brazilian locations was assembled and analyzed. A Bayesian phylogeographic generalized linear model approach was used to reconstruct the spatiotemporal history of HIV-1C in Brazil, considering several potential explanatory predictors of the viral diffusion process. Analyses were performed on several subsampled data sets in order to mitigate potential sample biases. We reveal a central role for the city of Porto Alegre, the capital of the southernmost state, in the Brazilian HIV-1C epidemic (HIV-1C_BR), and the northward expansion of HIV-1C_BR could be linked to source populations with higher HIV-1 burdens and larger proportions of HIV-1C infections. The results presented here bring new insights to the continuing discussion about the HIV-1C epidemic in Brazil and raise an alternative hypothesis for its spatiotemporal history. The current work also highlights how sampling bias can confound phylogeographic analyses and demonstrates the importance of incorporating external information to protect against this. IMPORTANCE: Subtype C is responsible for the largest HIV infection burden worldwide, but our understanding of its transmission dynamics remains incomplete. Brazil witnessed a relatively recent introduction of HIV-1C compared to HIV-1B, but it swiftly spread throughout the south, where it now circulates as the dominant variant. The northward spread has been comparatively slow, and HIV-1B still prevails in that region. While epidemiological data and viral genetic analyses have both independently shed light on the dynamics of spread in isolation, their combination has not yet been explored. Here, we complement publically available sequences and new genetic data from 13 cities with epidemiological data to reconstruct the history of HIV-1C spread in Brazil. The combined approach results in more robust reconstructions and can protect against sampling bias. We found evidence for an alternative view of the HIV-1C spatiotemporal history in Brazil that, contrary to previous explanations, integrates seamlessly with other observational data.

Detection of the B"-GWGR variant in the southernmost region of Brazil: unveiling the complexity of the human immunodeficiency virus-1 subtype B epidemic.

Typical human immunodeficiency virus-1 subtype B (HIV-1B) sequences present a GPGR signature at the tip of the variable region 3 (V3) loop; however, unusual motifs harbouring a GWGR signature have also been isolated. Although epidemiological studies have detected this variant in approximately 17-50% of the total infections in Brazil, the prevalence of B"-GWGR in the southernmost region of Brazil is not yet clear. This study aimed to investigate the C2-V3 molecular diversity of the HIV-1B epidemic in southernmost Brazil. HIV-1 seropositive patients were ana-lysed at two distinct time points in the state of Rio Grande do Sul (RS98 and RS08) and at one time point in the state of Santa Catarina (SC08). Phylogenetic analysis classified 46 individuals in the RS98 group as HIV-1B and their molecular signatures were as follows: 26% B"-GWGR, 54% B-GPGR and 20% other motifs. In the RS08 group, HIV-1B was present in 32 samples: 22% B"-GWGR, 59% B-GPGR and 19% other motifs. In the SC08 group, 32 HIV-1B samples were found: 28% B"-GWGR, 59% B-GPGR and 13% other motifs. No association could be established between the HIV-1B V3 signatures and exposure categories in the HIV-1B epidemic in RS. However, B-GPGR seemed to be related to heterosexual individuals in the SC08 group. Our results suggest that the established B"-GWGR epidemics in both cities have similar patterns, which is likely due to their geographical proximity and cultural relationship.

Bacteriophages as Potential Clinical Immune Modulators.

Bacteriophages (phages for short) are bacteria-specific viruses that have been drawing attention when it comes to countering the ever-growing antibiotic bacterial resistance, and are being seen as one of the most promising technologies against multi-antibiotic-resistant bacteria. Although bacteriophages are commonly regarded only as anti-bacterial objects unable to directly interact with eukaryotic cell metabolism, an increasing quantity of evidence has indicated that bacteriophages can directly affect cells bacteria in both in vitro and in vivo applications, influencing the behavior of tissues and immune systems. In sight of this new range of applications, several authors have expressed enthusiasm in phage therapy as direct modulators of eukaryotic cells for clinical usage, highlighting the need for further investigations covering the pharmacology of these new "eukaryotic-viruses", as even harmful interactions with eukaryotic cells were detected after phage therapy. The present review aims to cover and highlight mechanisms through which bacteriophages may interact with immune cells, analyzing potential clinical applications and obstacles presented in the use of bacteriophages as anti-inflammatory tools.

Age-dependent impairment in antibody responses elicited by a homologous CoronaVac booster dose.

The emergence of the SARS-CoV-2 Omicron sublineages resulted in increased transmission rates and reduced protection from vaccines. To counteract these effects, multiple booster strategies were used in different countries, although data comparing their efficiency in improving protective immunity remain sparse, especially among vulnerable populations, including older adults. The inactivated CoronaVac vaccine was among the most widely distributed vaccine worldwide and was essential in the early control of SARS-CoV-2-related hospitalizations and deaths. However, it is not well understood whether homologous versus heterologous booster doses in those fully vaccinated with CoronaVac induce distinct humoral responses or whether these responses vary across age groups. We analyzed plasma antibody responses from CoronaVac-vaccinated younger or older individuals who received a homologous CoronaVac or heterologous BNT162b2 or ChAdOx1 booster vaccine. All three evaluated boosters resulted in increased virus-specific IgG titers 28 days after the booster dose. However, we found that both IgG titers against SARS-CoV-2 Spike or RBD and neutralization titers against Omicron sublineages were substantially reduced in participants who received homologous CoronaVac compared with the heterologous BNT162b2 or ChAdOx1 booster. This effect was specifically prominent in recipients >50 years of age. In this group, the CoronaVac booster induced low virus-specific IgG titers and failed to elevate neutralization titers against any Omicron sublineage. Our results point to the notable inefficiency of CoronaVac immunization and boosting in mounting protective antiviral humoral immunity, particularly among older adults, during the Omicron wave. These observations also point to benefits of heterologous regimens in high-risk populations fully vaccinated with CoronaVac.

Effect of preexisting immunity to adenovirus on transgene product-specific genital T cell responses on vaccination of mice with a homologous vector.

We evaluated changes in global and human immunodeficiency virus (HIV)-specific genital T cells after vaccination of female mice with a replication-defective adenovirus vector of human serotype 5 (AdHu5) expressing Gag protein of HIV-1, in the presence or absence of preexisting immunity to the vaccine carrier. Our data show that preexisting immunity causes a rapid and transient decrease of genital CD4(+) T cells without increasing the expression of chemokine (C-C motif) receptor 5. Furthermore, preexposure to AdHu5 affects long-term alterations in the magnitude and quality of vaccine-induced Gag-specific CD8(+) T cell responses. AdHu5-specific antibodies interfere with the induction of transgene product-specific CD8(+) T cell responses in systemic compartments, whereas some mechanism other than antibodies also seems to affect those that home to the genital tract.

Prevalence and risk factors for HIV-1 infection in people who use illicit drugs in northern Brazil.

BACKGROUND: People who use illicit drugs (PWUDs) have a high risk of viral infections. To date, there is a paucity of information on HIV infection among PWUDs in remote Brazilian regions. This study determined the prevalence and factors associated with HIV-1 infection among PWUDs in northern Brazil. METHODS: Sociodemographic, economic, drug use and health-related information were collected through interviews from a community-recruited, multi-site sample of 1753 PWUDs. The blood samples collected were tested for the presence of HIV-1 using chemiluminescence immunoassay and PCR or western blotting. Logistic regressions identified factors independently associated with HIV-1 infection. RESULTS: In total, 266 (15.2%) PWUDs were HIV-1 positive. Hepatitis B virus and/or hepatitis C virus nucleic acid was detected in 65 (3.7%) PWUDs infected by HIV-1. The factors associated with HIV-1 infection were male gender, older age, a lower educational level and a lower income, crack cocaine use, a longer drug use history and a history of drug injection and engagement in unsafe sex, sex work and a higher number of sexual partners. CONCLUSIONS: The current study provides unique, initial insights into HIV and co-infection status and pertinent risk factors among PWUDs in northern Brazil, with clear and diverse implications for urgently improved prevention and treatment intervention needs.

Inferring population dynamics of HIV-1 subtype C epidemics in Eastern Africa and Southern Brazil applying different Bayesian phylodynamics approaches.

The subtype C Eastern Africa clade (CEA), a particularly successful HIV-1 subtype C lineage, has seeded several sub-epidemics in Eastern African countries and Southern Brazil during the 1960s and 1970s. Here, we characterized the past population dynamics of the major CEA sub-epidemics in Eastern Africa and Brazil by using Bayesian phylodynamic approaches based on coalescent and birth-death models. All phylodynamic models support similar epidemic dynamics and exponential growth rates until roughly the mid-1980s for all the CEA sub-epidemics. Divergent growth patterns, however, were supported afterwards. The Bayesian skygrid coalescent model (BSKG) and the birth-death skyline model (BDSKY) supported longer exponential growth phases than the Bayesian skyline coalescent model (BSKL). The BDSKY model uncovers patterns of a recent decline for the CEA sub-epidemics in Burundi/Rwanda and Tanzania (Re < 1) and a recent growth for Southern Brazil (Re > 1); whereas coalescent models infer an epidemic stabilization. To the contrary, the BSKG model captured a decline of Ethiopian CEA sub-epidemic between the mid-1990s and mid-2000s that was not uncovered by the BDSKY model. These results underscore that the joint use of different phylodynamic approaches may yield complementary insights into the past HIV population dynamics.

Higher cross-subtype IFN-γ ELISpot responses to Gag and Nef peptides in Brazilian HIV-1 subtype B- and F1- than in C-infected subjects.

HIV-1 diversity has been considered a huge challenge for the HIV-1 vaccine development. To overcome it, immunogens based on centralized sequences, as consensus, have been tested. In Brazil, the co-circulation of three subtypes offers a suitable scenario to test T cell cross-subtype responses to consensus sequences. Furthermore, we included peptides based on closest viral isolates (CVI) from each subtype analyzed to compare with T cell responses detected against the consensus sequences. The study included 32 subjects infected with HIV-1 subtype B (n=13),C (n=11), and F1 (n=8). Gag and Nef-specific T cell responses were evaluated by IFN-γ-ELISpot assay. Peptides based on CVI sequences were similar to consensus in both reducing genetic distance and detecting T cell responses. A high cross-subtype response between B and F1 in both regions was observed in HIV-1 subtype B and F1-infected subjects. We also found no significant difference in responses to subtype B and C consensus peptides among subtype B-infected subjects. In contrast, the magnitude of T cell responses to consensus C peptides in the Gag region was higher than to consensus B peptides among HIV-1 subtype C-infected subjects. Regarding Nef, subtype C-infected subjects showed higher values to consensus C than to consensus F1 peptides. Moreover, subtype F1-infected subjects presented lower responses to subtype C peptides than to subtype F1 and B. A similar level of responses was detected with group M based peptides in subtype B and F1 infected subjects. However, among subtype C infected subjects, this set of peptides detected lower levels of response than consensus C. Overall, the level of cross-subtype response between subtypes B and F1 was higher than between subtype C and B or C and F1. Our data suggests that the barrier of genetic diversity in HIV-1 group M for vaccine design may be dependent on the subtypes involved.

Novel monoclonal antibodies that bind to wild and fixed rabies virus strains.

Ten monoclonal antibodies (MAbs) against rabies virus, including IgG3κ, IgG2aκ, IgMκ, and an IgG2bκ isotype, were produced and characterized using neutralization, ELISA, immunodot-blot, and immunofluorescence assays. MAb 8D11, which recognized rabies virus glycoprotein, was found to neutralize rabies virus in vitro. When submitted to an immunofluorescence assay, seven MAbs showed different reactivity against 35 Brazilian rabies virus isolates. Three MAbs (LIA 02, 3E6, and 9C7) only failed to recognize one or two virus isolates, whereas MAb 6H8 was found to be reactive against all virus isolates tested. MAbs were also evaluated for their immunoreactivity against fixed rabies virus strains present in human and veterinary commercial vaccines. MAbs LIA 02, 6H8, and 9C7 reacted against all vaccine strains, while the remaining MAbs recognized at least 76% of vaccine strains tested. This research provides a set of MAbs with potential application for improving existing or developing new diagnostic tests and immunoassays.

Detection of the B"-GWGR variant in the southernmost region of Brazil: unveiling the complexity of the human immunodeficiency virus-1 subtype B epidemic

Typical human immunodeficiency virus-1 subtype B (HIV-1B) sequences present a GPGR signature at the tip of the variable region 3 (V3) loop; however, unusual motifs harbouring a GWGR signature have also been isolated. Although epidemiological studies have detected this variant in approximately 17-50% of the total infections in Brazil, the prevalence of B"-GWGR in the southernmost region of Brazil is not yet clear. This study aimed to investigate the C2-V3 molecular diversity of the HIV-1B epidemic in southernmost Brazil. HIV-1 seropositive patients were ana-lysed at two distinct time points in the state of Rio Grande do Sul (RS98 and RS08) and at one time point in the state of Santa Catarina (SC08). Phylogenetic analysis classified 46 individuals in the RS98 group as HIV-1B and their molecular signatures were as follows: 26% B"-GWGR, 54% B-GPGR and 20% other motifs. In the RS08 group, HIV-1B was present in 32 samples: 22% B"-GWGR, 59% B-GPGR and 19% other motifs. In the SC08 group, 32 HIV-1B samples were found: 28% B"-GWGR, 59% B-GPGR and 13% other motifs. No association could be established between the HIV-1B V3 signatures and exposure categories in the HIV-1B epidemic in RS. However, B-GPGR seemed to be related to heterosexual individuals in the SC08 group. Our results suggest that the established B"-GWGR epidemics in both cities have similar patterns, which is likely due to their geographical proximity and cultural relationship.

Molecular cloning and recombinant expression of the VP28 carboxyl-terminal hydrophilic region from a brazilian white spot syndrome virus isolate

In the present study, a fragment of the VP28 coding sequence from a Brazilian WSSV isolate (BrVP28) was cloned, sequenced and expressed in E. coli BL21(DE3) pLysS strain in order to produce the VP28 carboxyl-terminal hydrophilic region. The expression resulted in a protein of about 21 kDa, which was purified under denaturing conditions, resulting in a final highly purified BrVP28 preparation. The recombinant protein obtained can be used in several biotechnology applications, such as the production of monoclonal antibodies which could be used in the development of diagnostic tools as well as in the studies on the characterization of white spot syndrome virus (WSSV) isolated in Brazil.

Efeito da suplementação com L-alanil-L-glutamina sobre a resposta de hipersensibilidade do tipo tardio em ratos submetidos ao treinamento intenso / Effect of L-glutamine and L-alanyl-L-glutamine supplementation on the response to delayed-type hypersensitivity test (DTH) in rats submitted to intense training

O treinamento intenso e o exercício exaustivo podem ocasionar imunossupressão em atletas por meio da diminuição da concentração plasmática de glutamina. O presente estudo verificou inicialmente o efeito da suplementação com L-glutamina e L-alanil-L-glutamina sobre a resposta ao teste de hipersensibilidade do tipo tardio (HTT) em ratos submetidos ao treinamento intenso em natação durante seis semanas. Posteriormente, foi avaliado o efeito dessas intervenções nutricionais sobre a contagem total e porcentual de leucócitos e concentração sérica de anticorpos IgG anti-albumina de soro bovino, em animais submetidos ao teste de exaustão e recuperados durante o período de 3 horas...

Vacinaçäo de camundongos BALB/c com o gene da GP43 de Paracoccidioides brasiliensis: estudo da resposta imune / BALB/c mice vaccination with GP43 gene from Paracocidioides brasiliensis: study of imune response

Ácidos nucléicos têm sido utilizados como a terceira geração de vacinas, e têm sido utilizados em ensaios de imunoproteção contra infecções virais, bacterianas e parasitárias. Resultados prévios mostraram que a imunização com a gp43 nativa ou com um peptídeo de 15 aminoácidos (P1O) contido neste antígeno protegeu camundongos contra o desafio de leveduras virulentas de P. brasiliensis. Para determinar se similar imunidade protetora poderia ser conseguida pela imunização com DNA, a ORF da gp43 foi subclonada em um vetor de expressão para células de mamíferos denominado VR-1012 (Vical Inc.), criando assim o plasmídeo VR-gp43. Grupos de camundongos BALB/c foram imunizados com lOOmg de VR-gp43 ou VR-1012, num total de 4 doses, com intervalos de 2 semanas, por 2 meses. A resposta humoral contra a gp43 aumentou durante a imunização com VR-gp43, sendo IgG1, IgG2a, IgG2b e IgE os principais isótipos produzidos. Resposta imune celular também foi induzida, como medido pela estimulação ín vitro com gp43 de células de linfonodos dos animais imunizados com VR-gp43, com alta produção de IL-2 e IFN-Y. Estes resultados sugerem que uma resposta imune celular mista THllTH2, modulada por IFN-Y, foi induzida pela imunização com DNA neste modelo murino. A imunidade induzida foi duradoura e capaz de proteger camundongos BALB/c infectados com leveduras virulentas de P. brasiliensis, levando à significante diminuição dos UFCs nos pulmões e' redução ou não disseminação para baço e fígado dos camundongos, quando comparado com os animais controles. Estes resultados indicam que a imunização gênica e uma alternativa viável para induzir imunidade protetora também contra infecções fúngicas