Analysis of Immunological, Viral, Genetic, and Environmental Factors That Might Be Associated with Decreased Susceptibility to HIV Infection in Serodiscordant Couples in Florianópolis, Southern Brazil.

Individuals who have been exposed to human immunodeficiency virus (HIV) and have not been infected might possess natural resistance mechanisms. An understanding of the sociodemographic and immunological conditions that influence resistance to HIV is a challenge, and very little is known about the role of intrinsic antiviral factors that restrict HIV infection. The aim of this study was to analyze potential factors responsible for resistance to HIV infection in serodiscordant couples by comparing HIV-exposed seronegative individuals (HESN) to HIV-seropositive individuals treated with antiretroviral therapy (HIV-ART) along with healthy controls (HC). The results revealed one HLA-B*27 and two HLA-B*57 individuals among the HESN; a CCR5Δ32 heterozygous deletion was observed in one serodiscordant couple, while the homozygous genotype for this variant was not observed. There were no differences in the basal mRNA expression of APOBEC3G, CFLAR, TRIM5α, LEDGF/p75, BST-2, or SAMHD1 in CD4(+) T lymphocyte- and monocyte-enriched populations among the three groups, and lower HBD-3 concentrations were observed in saliva from HIV-ART compared to HESN and HC. The most prevalent HIV-1 subtype was C or C-containing recombinant forms. Six HIV-ART individuals and one HIV-ART individual were infected with the R5 HIV and X4 HIV strains, respectively. The ability to control infection or delay disease progression is probably defined by a balance between viral and host factors, and further evaluation should be performed in larger cohorts. Our data suggest that susceptibility to HIV infection varies among individuals and strengthens the multifactorial characteristics underlying the resistance mechanisms in HIV.

Immunophenotyping of classic murine myeloma cell lines used for monoclonal antibody production.

Murine myeloma cell lines play an important role in different areas of scientific research and are essential tools for monoclonal antibody production technology. Thus, it is important to understand the biology of these cell lines in order to provide useful information to various research fronts. The present study aims to perform detailed analyses of surface antigens expressed on three major murine myeloma cell lines extensively used for MAb production. The P3X63Ag8.653 cell line expresses molecules associated with T cell interaction (CD40(low), CD80(low)), as well as antigens related to plasma cell phenotype (CD138(high), CD184(low)). The Sp2/0-Ag14 cell line presents molecules associated with BCR activation and regulation (CD79b(low), CD22(low), CD72(med)), molecules related to T cell interaction (CD40(low), CD80(low)), and markers of plasma cell phenotype (CD138(high), CD184(low)). The NS1 cell line presents all molecules of plasma cell phenotype evaluated in this study (CD184(low), CD138(high), CD38(med)) with low expression of CD72 (CD72(low)), a molecule related to BCR activation. Molecules associated with immune response modulation such as CD23 and CD25, as well as CD117, a marker related to undifferentiated cell phenotype, were not observed in any of the three murine myeloma cell lines evaluated. These data show that in spite of their common origin and function, the immunological profiles differ between P3X63Ag8.653, Sp2/0-Ag14, and NS1 cell lines.

Detection of infectious myonecrosis virus in penaeid shrimps using immunoassays: usefulness of monoclonal antibodies directed to the viral major capsid protein.

Despite the economic impact of the infectious myonecrosis virus (IMNV) on shrimp farms in several countries, no method for immunological detection is currently available. With the aim of developing immunodiagnostic methods for IMNV detection in infected shrimps, a recombinant fragment of the IMNV major capsid protein gene encoding amino acids 105-297 (rIMNV105₋297 was heterologously expressed in Escherichia coli and used to immunize Balb/c mice, generating monoclonal antibodies (MAbs). Six hybridomas were obtained, and four of these recognized the presence of IMNV in tissue homogenates from naturally infected shrimps by immunodot blot assay. Among these MAbs, three were able to detect a ~100-kDa protein, which corresponds to the predicted mass of the IMNV major capsid protein, as well as viral inclusion bodies in muscle fibroses by western blot and immunohistochemistry. Two MAbs showed high specificity and sensitivity, showing no cross-reaction with healthy shrimp tissues in any assays, indicating their usefulness for IMNV detection.

Neutralizing antibodies to human and simian adenoviruses in humans and New-World monkeys.

Vaccines based on adenovirus (Ad) vectors are currently in development against several pathogens. However, neutralizing antibodies (NAb) to human adenovirus type 5 (AdHu5), the best-studied vector, are highly prevalent in humans worldwide. Less-prevalent adenoviruses, including human and simian serotypes, provide alternative vaccine platforms. In this study, sera from 200 Brazilian human subjects and New-World monkeys were tested for NAb titers to human serotypes AdHu5 and AdHu26 and chimpanzee-origin Ad viruses of serotype 6 (AdC6) and serotype 68 (AdC68). Seroprevalence rates of NAb in humans were 69.5% for AdHu5, 44% for AdHu26, 21% for AdC6 and 23.5% for AdC68. In addition, NAb titers to human Ad were consistently higher than those found to simian serotypes. Surprisingly, sera from some New-World monkey species were able to neutralize AdC6 and/or AdC68. A possible explanation for these findings and the implications for the development of Ad-vector vaccines are discussed in detail.

Detection of major capsid protein of infectious myonecrosis virus in shrimps using monoclonal antibodies.

Infectious myonecrosis virus (IMNV) has been causing a progressive disease in farm-reared shrimps in Brazil and Indonesia. Immunodiagnostic methods for IMNV detection, although reliable, are not employed currently because monoclonal antibodies (MAbs) against this virus are not available. In this study, a fragment of the IMNV major capsid protein gene, comprising amino acids 300-527 (IMNV(300-527)), was cloned and expressed in Escherichia coli. The nucleotide sequence of the recombinant IMNV(300-527) fragment displayed a high degree of identity to the major capsid protein of IMNV isolates from Brazil (99%) and Indonesia (98%). Ten MAbs were generated against the expressed fragment, and eight of these, mostly IgG(2a) or IgG(2b), were able to bind to IMNV in tissue extracts from shrimps infected naturally in immunodot-blot assays. Six of these MAbs recognized a approximately 100 kDa protein in a Western-blot, which is the predicted mass of IMNV major capsid protein, and also bound to viral inclusions present in muscle fibroses and in coagulative myonecrosis, as demonstrated by immunohistochemistry. Among all those MAbs created, four did not cross-react with non-infected shrimp tissues; this observation supports their applicability as a sensitive and specific immunodiagnosis of IMNV infection in shrimps.

A novel monoclonal antibody that binds to hemocytes from shrimps and oysters.

The monoclonal antibody (MAb) LITO-1 was produced from a stable hybridoma cell line generated by the fusion of NS1 myeloma cells with spleen cells isolated from Balb/c mice immunized with a paraformaldehyde-fixed hemocyte suspension of Litopenaeus vannamei. This MAb reacted with all three hemocyte subtypes, but no reaction was observed with components of plasma. Immunohistochemistry assays demonstrated that LITO-1 was very effective in specifically distinguishing hemocytes infiltrated in several tissues such as striated muscle, brain, and hepatopancreas. Moreover, this antibody was able to recognize hemocytes from two shrimp species, Litopenaeus schmitti and Farfantepenaeus paulensis, as well as hemocytes of the oyster Crassostrea gigas. No reaction was observed against hemocytes from the terrestrial insect Triatoma klugi or with mammalian RAW cells. This novel MAb can be useful in revealing the presence and function of a conservative epitope in hemocytes of marine crustaceans and mollusks.

Production and characterization of monoclonal antibodies against the recombinant nucleoprotein of Araucaria hantavirus.

Hantaviruses are rodent-borne RNA viruses that cause hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). From the first detection of infection in Brazil in 1993 until 2009, 1161 cases of HPS have been reported, with mortality rates of around approximately 40%. Currently, due to the absence of a vaccine or specific antiviral therapy, the only way to reduce mortality by hantavirus infection is a fast and precise diagnosis that allows for supportive clinical health care. To improve the detection of hantavirus infection, we developed monoclonal antibodies (Mabs) against the nucleoprotein (rNDelta85) of the Araucaria hantavirus strain (ARAUV). The specificity of generated Mabs for rNDelta85 was demonstrated by western blot, indirect immunofluorescence and immunohistochemistry. These are the first monoclonal antibodies to be produced and characterized against the South American hantavirus strain, and may be of special interest in the development of diagnostic assays and epidemiological studies.

Therapeutic monoclonal antibodies in ophthalmology.

Monoclonal antibodies (mAbs) can be used therapeutically by binding to molecular targets with high specificity. Therefore, they have excellent therapeutic applications in ophthalmology. This manuscript presents four aspects of the therapeutic use of mAbs in ophthalmology: the scientific rationale, the unique characteristics of selected mAbs, the current state-of-the-art application, and relevant therapeutic mAbs for future applications in ophthalmology. We identified in the literature various single-agent therapies that inhibit the following targets: tumor necrosis factor (TNF), epithelial growth factor receptor, vascular endothelial growth factor (VEGF) receptor, basic fibroblast growth factor receptor, platelet-derived growth factor, and cluster of differentiation antigens. The roles of all biochemical targets in ocular diseases were evaluated. Current and future mAbs against various cytokines were assessed for the treatment of ocular diseases. The medical literature showed the clinical benefits of mAbs for treating angiogenic and inflammatory ocular diseases. Two anti-VEGF mAbs, bevacizumab and ranibizumab, and three anti-TNF agents, infliximab, etanercept, and adalimumab, control ocular neovascularization and intraocular inflammation. Other mAbs such as rituximab, daclizumab, efalizumab, and alemtuzumab showed positive results in animal and early clinical studies and may represent useful adjuvant therapies for ocular lymphoma or ocular inflammation. Ranibizumab is the only FDA-approved therapy; for other mAbs the so-called off-label application remains the standard. Intravenous administration of mAbs has demonstrated acceptable toxicity profiles, while intraocular injection may decrease the chances of systemic complications and increase the amount of drug available to the retina and choroid. In conclusion, effective clinical use of mAbs in ophthalmology is more commonly seen in the field of angiogenic vitreoretinal and autoimmune inflammatory diseases. The challenge for the future is combining biologic therapies to improve the quality and duration of responses while diminishing side effects. The role of mAbs within ophthalmic treatments will be defined according to future clinical experience and the results of randomized clinical trials.

Expression, purification and immunodetection of a recombinant fragment (residues 179-281) of the G protein from rabies virus ERA strain.

The glycoprotein (G) of rabies virus (RV) is important for virus infectivity and induction of the protective immunity. In this study, the region comprising linear epitopes (residues 179-281, ERA strain), named rGERA179-281, was cloned in frame with a hexahistidine tag coding sequence at its N-terminal end and overexpressed in Escherichia coli Rosetta strain. The expression under transcriptional regulation of T7 promoter yielded insoluble protein aggregates in the form of inclusion bodies. The inclusion bodies were solubilized with 6M guanidine HCl and the protein was purified to homogeneity under denaturing conditions. Mass spectrometry data confirmed the identity of the protein. The purified protein (13.8kDa) showed significant reactivity with antibodies present in a therapeutic human rabies immune globulin (HRIG), as demonstrated by immunoblotting analysis. In addition, by in vitro competitive neutralization assay, rGERA179-281 led to a measurable reduction in the ability of HRIG to neutralize rabies virus. These results, along with the good yield obtained, encourage further studies on the more detailed immunological properties of rGERA179-281, such as the ability to induce rabies virus neutralizing antibodies and the production of anti-G monoclonal antibodies, which together, might be useful for the development of new diagnostic methods.

An optimized nested polymerase chain reaction (PCR) approach allows detection and characterization of human immunodeficiency virus type 1 (HIV-1) env and gag genes from clinical samples.

The needs for development and/or improvement of molecular approaches for microorganism detection and characterization such as polymerase chain reaction (PCR) are of high interest due their sensitivity and specificity when compared to traditional microbiological techniques. Considering the worldwide importance of human immunodeficiency virus type 1 (HIV-1) infection, it is essential that such approaches consider the genetic variability of the virus, the heterogeneous nature of the clinical samples, the existence of contaminants and inhibitors, and the consequent needs for standardization in order to guarantee the reproducibility of the methods. In this work we describe a nested PCR assay targeting HIV-1 virus gag and env genes, allowing specific and sensitive diagnosis and further direct characterization of clinical samples. The method described herein was tested on clinical samples and allowed the detection of HIV-1 presence in all samples tested for the gag gene and 90.9% for the env gene, revealing sensitivities of 1 fg and 100 fg, respectively. Also, no cross-reactions were observed with DNA from infected and noninfected patients and the method allowed detection of the env and gag genes on an excess of 10(8) and 10(4) of human deoxyribonucleic acid (DNA), respectively. Furthermore, it was possible to direct sequence all amplified products, which allowed the sub typing of the virus in clinical samples.

Molecular epidemiology of HIV-1 in Santa Catarina State confirms increases of subtype C in Southern Brazil.

Recent studies have demonstrated an increased prevalence of human immunodeficiency virus type 1 (HIV-1) subtype C in southern Brazil. Although Santa Catarina State (SC) is located in this area and presents one of the country's highest incidences of HIV/AIDS, knowledge on the molecular epidemiology of HIV-1 in such State is lacking. The aim of this study was to investigate the HIV-1 molecular diversity and epidemiological profile of HIV-1-infected patients from SC. DNA samples were PCR amplified and HIV-1 subtypes were determined using both env and gag genes by direct sequencing. Phylogenetic analyses revealed that 48% were subtype C and 23% were subtype B. Possible recombinant forms were observed for both B/C (23%) and B/F (6%) subtypes. Our results, for the first time, identifies HIV-1 subtype C as a major clade circulating in SC and contributes to the understanding of HIV epidemics in the country by confirming the epidemic spread of the HIV-1 subtype C in southern Brazil.

Evaluation of antiviral activity of South American plant extracts against herpes simplex virus type 1 and rabies virus.

This paper describes the screening of different South American plant extracts and fractions. Aqueous and organic extracts were prepared and tested for antiherpetic (HSV-1, KOS and 29R strains) and antirabies (PV strain) activities. The evaluation of the potential antiviral activity of these extracts was performed by using an MTT assay for HSV-1, and by a viral cytopathic effect (CPE) inhibitory method for rabies virus (RV). The results were expressed as 50% cytotoxicity (CC(50)) for MTT assay and 50% effective (EC(50)) concentrations for CPE, and with them it was possible to calculate the selectivity indices (SI = CC(50)/EC(50)) of each tested material. From the 18 extracts/fractions tested, six extracts and four fractions showed antiviral action. Ilex paraguariensis, Lafoensia pacari, Passiflora edulis, Rubus imperialis and Slonea guianensis showed values of SI > 7 against HSV-1 KOS and 29-R strains and Alamanda schottii showed a SI of 5.6 against RV, PV strain.

Genital CD8+ T cell response to HIV-1 gag in mice immunized by mucosal routes with a recombinant simian adenovirus.

AdC6gag37, an E1-deleted adenovirus recombinant derived from the chimpanzee adenovirus serotype 6 expressing a codon-optimized truncated form of gag of HIV-1, was tested for induction of transgene-specific CD8+ T cell responses upon intranasal or intravaginal immunization of mice. Administration of AdC6gag37 induced gag-specific CD8+ T cells at systemic and mucosal sites. Frequencies of gag-specific CD8+ T cells elicited in the genital tract by intravaginal or intranasal immunizations were substantially increased by intranasal priming followed by intravaginal boosting with the same vector. Additionally, intravaginal immunization with AdC6gag37 increased the amount of gammadelta T cells that could be detected in genital tract.

Evaluation of antiviral activity of phenolic compounds and derivatives against rabies virus.

Human rabies is a viral disease with a great impact on public health, mainly on account of its fatal course in the majority of cases. Despite the well-established prophylaxis by immunization, rabies is believed to be responsible for 40,000-70,000 human deaths per year, mostly in endemic areas. Palliative support and experimental protocols to avoid death have been employed with no expressive results, with the exception of a recent human case of recovery from rabies. No antiviral drugs are currently available to fight against this infection. In combination with the prophylaxis, an antiviral drug would be useful for human rabies treatment, providing enhanced protection against the encephalitis caused by the virus. Phenolic compounds are derived from the secondary plant metabolism, although they can also be obtained by synthetic processes. Many studies have shown a great range of pharmacological effects for these substances, including vasodilatation, antiallergenic, antiinflammatory and antiviral properties, among others. In this study, the potential in-vitro anti-rabies activity of 24 synthetic phenolic compounds was evaluated using McCoy cells and PV rabies strain. The cytotoxicity (CC50) was assayed by the MTT method and the antiviral activity (IC50) was estimated by the inhibition of viral cytopathic effects. Isoprinosine and ketamine were used as positive controls. The tested compounds showed selectivity indices (SI=CC50/IC50) ranging from 1.0 to 3.9. Six phenolic compounds failed to inhibit the cytopathic effect to any degree, and four showed SI > or = 3.0. According to these results, some probable structure-activity relationships are suggested. It was observed that the presence of free hydroxyl and ether groups influenced the anti-rabies activity. However, additional studies are required to establish these relationships.

Serial changes in plasma levels of cytokines in patients with coronary artery disease.

OBJECTIVES: Inflammation is known to be a major determinant of the progression of coronary artery disease (CAD). In the present study we have evaluated the plasma levels of cytokines-tumor necrosis factor-alpha (TNF), interleukin- 1 alpha (IL-1), interleukin-6 (IL-6), interferon-gamma (IFN), and interleukin-10 (IL-10)- to examine the association between these cytokines and C-reactive protein (CRP) in patients with CAD. METHODS: Patients with acute coronary syndromes (ACS; n = 20) were compared with patients with stable angina (SA; n= 20) and with control volunteers (C; n= 20). Blood samples were collected at the time of admission from all patients and 15 and 30 days thereafter. RESULTS: CRP levels (20.8+/-8.8 mg/L) (mean+/-SEM) were higher at baseline in ACS than SA patients (4.1+/-0.8mg/L) or the control subjects (5.1+/-1.8mg/L) (p<0.05). At admission, IL-6 was detected in 50% of the ACS patients and 5% of the SA patients or control subjects, while TNF was detected in 35% of the ACS and SA patients but only in 5% of control subjects. Subsequently, IL-6 levels declined and were no longer detectable, while TNF levels increased among ACS patients at all time periods tested when compared with other patients. The presence of IL-1 and IL-10 were not detectable in the blood samples examined, and IFN could only be detected in the ACS group. A significant correlation was observed between IL-6 and CRP levels (r=0.4; p<0.01) in all groups. There were no correlations among any of the other cytokines and CRP levels. CONCLUSIONS: Our study demonstrates raised levels of TNF, IL-6, IFN, and CRP in patients with ACS and a positive correlation between IL-6 and CRP but not with the other cytokines.

Adjuvantes de vacinas: possibilidades de uso em seres humanos ou animais / Vacine adjuvants: possibilities for use in humans or animals

Objetivo: Rever de forma sistemática as substânciasadjuvantes mais estudadas, utilizadas e as novassubstâncias; com o intuito de atualizar os conceitos desua ação por atuarem, juntamente com o antígeno, nosistema imunológico e seu futuro uso em vacinas humanasou animais. Métodos: Foi realizada revisão sistemática em livros e da literatura publicada no Medline® a partir do ano de 1969. Foram selecionados artigos que abordassemo tema “adjuvantes” nos seguintes aspectos: Respostainflamatória, resposta imune celular, resposta imune humoral, seqüelas, proteção. Resultados: Foram selecionados artigos originais ou capítulos de livros abordando este tema. Observou-se haver diferenças na capacidade de indução inflamatória entre os diferentes adjuvantes. Estas diferenças podem resultar em padrões de resposta imune celular e humoral diferenciados, de acordo com a célula mais estimulada. Assim, adjuvantes indutores de migração predominantemente mononuclear parecem facilitar respostas do padrão celular e humoral. Respostas mais neutrofílicas podem resultar em maiores efeitos colaterais locais e/ou sistêmicos e também podemser associados à redução da disponibilidade antigênicapara o sistema imunológico. Conclusões: O sistema imunológico animal ou humano pode se comportar de maneira diferenciada frente aos diferentes tipos de substâncias adjuvantes. Os mesmos mecanismos usados pelo organismo para proteção contra infecções podem, em situações particulares, serem causadores de doenças, por isso a importância do estudo individual de cada adjuvante a ser utilizado em cada situação e a necessidade de se encontrar uma substância que seja efetiva frente a grande número de antígenos e que diminua sobremaneira possíveis seqüelas.

Engano na administração de soro-vacinação para raiva. Emprego de esquema improvisado e sua avaliação: apresentação de um caso / An error in the administration of rabies serum vaccination. The use of an improvised schedule and its evaluation: report of a case

We report on a female patient attacked by a dog that died 4 days later, who sought treatment 11 days after the accident. A serum vaccination schedule was indicated, to be started immediately with the administration of anti-rabies serum (9 ml, corresponding to 40 IU/Kg body weight) and a series of 10 doses of vaccine applied on consecutive days plus 3 booster doses applied at 10-day intervals, according to the regulations of the Health Secretariat of the State of São Paulo. However, due to an error, 9 vaccine doses were initially applied at 3 different anatomical sites. The error was immediately discovered and it was decided to interrupt treatment for a few days and to restart and complete it later; this was done only 8 days later. Serologic follow-up by the serum-neutralization test in cell culture revealed a fully satisfactory response greatly exceeding WHO recommendations in terms of levels, precocity and duration. The patient continued to be healthy by the 240th day after the accident, when she was observed for the last time before this publication.

Immunomodulatory effect of glucan on the response to experimental antirabies vaccination

The objective of the present study was to determine the stimulatory response to antirabies vaccination promoted by glucan in mice. Glucan increased both resistance to infection and antibody titres and this effect was more evident when glucan was used at dose of 0.5 mg, administered intraperitoneally before, during and after immunization and when the challenge virus was applied to the foot-pad.