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ABSTRACT: 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.
Hybridoma (2005) 02/2012; 31(1):1-6. · 0.42 Impact Factor
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ABSTRACT: The HIV-1 subtype C has spread efficiently in the southern states of Brazil (Rio Grande do Sul, Santa Catarina and Paraná). Phylogeographic studies indicate that the subtype C epidemic in southern Brazil was initiated by the introduction of a single founder virus population at some time point between 1960 and 1980, but little is known about the spatial dynamics of viral spread. A total of 135 Brazilian HIV-1 subtype C pol sequences collected from 1992 to 2009 at the three southern state capitals (Porto Alegre, Florianópolis and Curitiba) were analyzed. Maximum-likelihood and Bayesian methods were used to explore the degree of phylogenetic mixing of subtype C sequences from different cities and to reconstruct the geographical pattern of viral spread in this country region. Phylogeographic analyses supported the monophyletic origin of the HIV-1 subtype C clade circulating in southern Brazil and placed the root of that clade in Curitiba (Paraná state). This analysis further suggested that Florianópolis (Santa Catarina state) is an important staging post in the subtype C dissemination displaying high viral migration rates from and to the other cities, while viral flux between Curitiba and Porto Alegre (Rio Grande do Sul state) is very low. We found a positive correlation (r(2) = 0.64) between routine travel and viral migration rates among localities. Despite the intense viral movement, phylogenetic intermixing of subtype C sequences from different Brazilian cities is lower than expected by chance. Notably, a high proportion (67%) of subtype C sequences from Porto Alegre branched within a single local monophyletic sub-cluster. These results suggest that the HIV-1 subtype C epidemic in southern Brazil has been shaped by both frequent viral migration among states and in situ dissemination of local clades.
PLoS ONE 01/2012; 7(4):e35649. · 4.09 Impact Factor
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ABSTRACT: The AIDS epidemic in Southern Brazil has unique features, showing co-circulation of HIV-1 subtypes C, B and recombinant forms. Florianópolis has the second highest AIDS incidence among Brazilian capitals, but limited information is available about HIV molecular epidemiology and prevalence of primary drug resistance.
To investigate the molecular epidemiology of HIV-1 in Florianópolis and to describe the prevalence of primary HIV-1 drug resistance mutations (DMRs).
Epidemiological and clinical data from 82 untreated patients from Florianópolis (2008-2009) were analyzed. The HIV-1 subtype at envelope, protease, reverse transcriptase and integrase regions were determined by phylogenetic and bootscaning analyses and the drug resistance profile were analyzed at the Stanford HIV Drug Resistance Database.
The most frequent HIV-1 genetic form was subtype C (65.8%) followed by mosaics BC (18.3%), subtype B (13.4%), subtype F1 (1.2%) and BCF1 recombinant (1.2%). HIV-1 subtype C and BC recombinants were much more frequent in the heterosexual exposure category, whereas subtype B was more common in the MSM exposure category. DRMs were seen in 11% of the sequences, 2.4% of them were related to PI, 5% to NRTI, 3.6% to NNRTI and 1.2% was related to INTI.
The present study confirms the high prevalence of subtype C and BC recombinants in Santa Catarina State and revealed a significant difference in the subtype distribution among distinct virus exposure categories. This study also shows a relative high prevalence of protease/reverse transcriptase primary drug resistance mutations and corroborates the usefulness of the integrase inhibitors in southern Brazil.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 07/2011; 51(3):186-91. · 3.12 Impact Factor
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ABSTRACT: Most studies on E1-deleted adenovirus (Ad) vectors as vaccine carriers for antigens of HIV-1 have focused on induction of central immune responses, although stimulation of mucosal immunity at the genital tract (GT), the primary port of entry of HIV-1, would also be highly desirable. In this study, different immunization protocols using chimpanzee-derived adenoviral (AdC) vectors expressing Gag of HIV-1 clade B given in heterologous prime-boost regimens were tested for induction of systemic and genital immune responses. Although i.n. immunization stimulated CD8(+) T-cell responses that could be detected in the GT, this route induced only marginal cellular responses in systemic tissues and furthermore numbers of Gag-specific CD8(+) T cells contracted sharply within a few weeks. On the contrary, i.m. immunization induced higher and more sustained frequencies of vaccine-induced cells which could be detected in the GT as well as systemic compartments. Antigen-specific CD8(+) T cells could be detected 1 year after immunization in all compartments analyzed. Genital memory cells secreted IFN-γ, expressed high levels of CD103 and their phenotypes were consistent with a state of activation. Taken together, the results presented here show that i.m. vaccination with chimpanzee-derived (simian) adenovirus vectors is a suitable strategy to induce a long-lived genital CD8(+) T-cell response.
European Journal of Immunology 12/2010; 40(12):3426-38. · 5.10 Impact Factor
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ABSTRACT: 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.
Virology 11/2010; 407(1):1-6. · 3.35 Impact Factor
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ABSTRACT: 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.
Journal of virological methods 10/2010; 169(1):169-75. · 2.13 Impact Factor
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ABSTRACT: 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.
Archives of Virology 09/2010; 156(1):9-16. · 2.11 Impact Factor
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ABSTRACT: Although it has been demonstrated that mucosal immunization using vectors such as simian adenovirus (AdC) stimulates robust adaptive immune responses, there remains a paucity of information on the modulation of innate immune responses by such vectors. Using an established murine model of intravaginal immunization (Ivag), we have investigated whether mucosal gammadelta T cells participate in immune responses induced by AdC vectors. gammadelta T cell numbers were found to be increased in the vaginal tract. Moreover, gammadelta T cells isolated from the genital tract showed an activated phenotype and enhanced expression of cytokine gene. Altogether, our results demonstrate that AdC modulates gammadelta T cell responses and suggest that this cell population may influence immune responses following vaginal immunization.
Vaccine 06/2010; 28(29):4600-8. · 3.77 Impact Factor
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ABSTRACT: 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.
Hybridoma (2005) 04/2010; 29(2):161-7. · 0.42 Impact Factor
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ABSTRACT: Although the development of an effective HIV-1 vaccine has proved very challenging for more than two decades, it remains the best hope to control the HIV pandemic. Since Brazil has particular epidemiological features, as well as adequate policies and infrastructure, the country has been an interesting site for HIV vaccine trials. Since 1995, eight trials were performed in Brazil enrolling over 2000 subjects. Peptide vaccine candidates were initially designed to elicit neutralising antibodies as an attempt to provide sterilising immunity against HIV-1. This strategy, however, has proved extremely difficult, and candidates were poorly immunogenic. Therefore, the next vaccine candidates focused mainly on the induction of cell mediated immune responses that would limit AIDS progression and transmission by suppressing viremia. Such candidates were naked DNA or viral vectors in either prophylactic or therapeutic approaches. Even though several candidates were immunogenic, protective immune responses against HIV-1 remain to be achieved. However, several studies with non-human primates and human elite controllers demonstrate that effective immune responses against HIV-1 may be elicited, supporting the belief that an HIV-1 vaccine is possible. Much has been learned, and now the development of an effective HIV-1 vaccine requires resetting priorities with focus on basic research, considering the merits of neutralising antibodies and CMI, as well as the role of innate immunity on HIV-1 protection. In this new perspective, large-scale trials should be replaced by smaller preliminary efficacy studies.
Reviews in Medical Virology 09/2009; 19(5):301-11. · 7.20 Impact Factor
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ABSTRACT: 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.
Journal of Clinical Laboratory Analysis 02/2008; 22(2):106-13. · 1.38 Impact Factor
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ABSTRACT: 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.
Journal of Medical Virology 11/2007; 79(10):1455-63. · 2.82 Impact Factor
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ABSTRACT: 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. J. Med. Virol. 79:1455–1463, 2007. © Wiley-Liss, Inc.
Journal of Medical Virology 09/2007; 79(10):1455 - 1463. · 2.82 Impact Factor
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ABSTRACT: 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.
Vaccine 02/2007; 25(1):109-16. · 3.77 Impact Factor
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ABSTRACT: 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.
Veterinary Microbiology 09/2006; 116(1-3):53-9. · 3.33 Impact Factor
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ABSTRACT: 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 ≥ 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.
Veterinary Microbiology.
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ABSTRACT: Pinto AR & Ertl HC (2002). Genetically modified adenoviruses as recombinant vaccines. Curr Topics in Virology, 2: 70-84. 1 ABSTRACT Recombinant adenoviruses have been tested extensively for their potential use as vaccines. They can be engineered to express heterologous proteins, which they present very efficiently to the immune system. Recombinant E1-deleted adenoviruses are safe due to their defect in replication. The comparatively small size of the viral genome furthermore allows for construction of recombinant adenoviruses derived from so-called molecular clones that are propagated in form of plasmid vectors in a bacterial host. This eliminates any risk of an inadvertent contamination with an unknown pathogenic entity that can otherwise readily escape detection if vaccine viruses are exclusively grown in mammalian cell lines. E1-deleted adenoviruses induce excellent immunity upon application to mucosal membranes, suggesting their usefulness as vaccines for infectious agents that enter through the airways, the mucosa of the gut or the genital tract. In the past 15 years, recombinant adenoviruses expressing a variety of antigens have been constructed and tested. Based on the encouraging results, several clinical studies have been undertaken. These promising results are now leading to improved protocols such as prime-boost regimes to elicit broader pathogen-specific cellular and humoral immune responses. Thus far, mainly E1-deleted adenoviral recombinants derived from common human serotypes have been employed as experimental vaccines. Such recombinants have one disadvantage: most humans are naturally infected with these viruses first during childhood and then periodically thereafter. The ensuing virus neutralizing antibodies against structural proteins of adenovirus most notably against the hexon impair the efficacy of adenoviral vaccines based on the homologous serotype. This can be circumvented by the recent development of adenoviral recombinants based on serotypes derived from other species that are closely related to humans such as chimpanzees. This article reviews the main perspectives of adenoviral recombinants that render them attractive for vaccine development.
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ABSTRACT: 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 γδ T cells that could be detected in genital tract.
Vaccine.
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ABSTRACT: Although it has been demonstrated that mucosal immunization using vectors such as simian adenovirus (AdC) stimulates robust adaptive immune responses, there remains a paucity of information on the modulation of innate immune responses by such vectors. Using an established murine model of intravaginal immunization (Ivag), we have investigated whether mucosal γδ T cells participate in immune responses induced by AdC vectors. γδ T cell numbers were found to be increased in the vaginal tract. Moreover, γδ T cells isolated from the genital tract showed an activated phenotype and enhanced expression of cytokine gene. Altogether, our results demonstrate that AdC modulates γδ T cell responses and suggest that this cell population may influence immune responses following vaginal immunization.
Vaccine.
