Control of human immunodeficiency virus type-1 protease activity in insect cells expressing Gag-Pol rescues assembly of immature but not mature virus-like particles

Expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in insect cells using baculovirus vectors leads to the abundant production of virus-like particles (VLPs) that represent the immature form of the virus. When Gag-Pol is included, however, VLP production is abolished, a result attributed to premature protease activation degrading the intracellular pool of Gag precursor before particle assembly can occur. As large-scale synthesis of mature noninfectious VLPs would be useful, we have sought to control HIV protease activity in insect cells to give a balance of Gag and Gag-Pol that is compatible with mature particle formation. We show here that intermediate levels of protease activity in insect cells can be attained through site-directed mutagenesis of the protease and through antiprotease drug treatment. However, despite Gag cleavage patterns that mimicked those seen in mammalian cells, VLP synthesis exhibited an essentially all-or-none response in which VLP synthesis occurred but was immature or failed completely. Our data are consistent with a requirement for specific cellular factors in addition to the correct ratio of Gag and Gag-Pol for assembly of mature retrovirus particles in heterologous cell types. (C) 2003 Elsevier Science (USA). All rights reserved.

Antiretroviral resistance mutations in human immunodeficiency virus type 1 infected patients enrolled in genotype testing at the Central Public Health Laboratory, São Paulo, Brazil: preliminary results

Antiretroviral resistance mutations (ARM) are one of the major obstacles for pharmacological human immunodeficiency virus (HIV) suppression. Plasma HIV-1 RNA from 306 patients on antiretroviral therapy with virological failure was analyzed, most of them (60%) exposed to three or more regimens, and 28% of them have started therapy before 1997. The most common regimens in use at the time of genotype testing were AZT/3TC/nelfinavir, 3TC/D4T/nelfinavir and AZT/3TC/efavirenz. The majority of ARM occurred at protease (PR) gene at residue L90 (41%) and V82 (25%); at reverse transcriptase (RT) gene, mutations at residue M184 (V/I) were observed in 64%. One or more thymidine analogue mutations were detected in 73%. The number of ARM at PR gene increased from a mean of four mutations per patient who showed virological failure at the first ARV regimens to six mutations per patient exposed to six or more regimens; similar trend in RT was also observed. No differences in ARM at principal codon to the three drug classes for HIV-1 clades B or F were observed, but some polymorphisms in secondary codons showed significant differences. Strategies to improve the cost effectiveness of drug therapy and to optimize the sequencing and the rescue therapy are the major health priorities.

Detection of immunoglobulin G antibodies to Neospora caninum in humans: High seropositivity rates in patients who are infected by human immunodeficiency virus or have neurological disorders

Considering that little is known about the epidemiology of Neospora caninum infection in humans, particularly in populations with high Toxoplasma gondii infection rates, the present study aimed to investigate the presence of antibodies to N. caninum in T. gondii-seropositive and -seronegative individuals. A total of 256 serum samples divided into four groups (61 samples from human immunodeficiency virus [HIV]-positive patients, 50 samples from patients with neurological disorders, 91 samples from newborns, and 54 samples from healthy subjects) were assessed for N. caninum and T. gondii serologies by indirect fluorescent-antibody test, enzyme-linked immunosorbent assay, and immunoblotting (IB). Immunoglobulin G antibodies to N. caninum were predominantly detected in HIV-infected patients (38%) and patients with neurological disorders (18%), while newborns and healthy subjects showed lower seropositivity rates (5% and 6%, respectively). Seropositivity to N. caninum was significantly associated with seropositivity to T. gondii in both HIV-infected patients and patients with neurological disorders. Seroreactivity to N. caninum was confirmed by IB, with positive sera predominantly recognizing the 29-kDa antigen of N. caninum. The results of this study indicate the presence of N. caninum infection or exposure in humans, particularly in HIV-infected patients or patients with neurological disorders, who could have opportunistic and concurrent infections with T. gondii. These findings may bring a new concern for the unstable clinical health of HIV-infected patients and the actual role of N. caninum infection in immunocompromised patients.

Frequencies of CCR5-D32, CCR2-64I and SDF1-3'A mutations in Human Immunodeficiency Virus (HIV) seropositive subjects and seronegative individuals from the state of Pará in Brazilian Amazonia

ABSTRACT: The distribution of genetic polymorphisms of chemokine receptors CCR5-D32, CCR2-64I and chemokine (SDF1-3 A) mutations were studied in 110 Human Immunodeficiency Virus type 1 (HIV-1) seropositive individuals (seropositive group) and 139 seronegative individuals (seronegative group) from the population of the northern Brazilian city of Belém which is the capital of the state of Pará in the Brazilian Amazon. The CCR5-D32 mutation was found in the two groups at similar frequencies, i.e. 2.2% for the seronegative group and 2.7% for the seropositive group. The frequencies of the SDF1-3 A mutation were 21.0% for the seronegative group and 15.4% for the seropositive group, and the CCR2-64I allele was found at frequencies of 12.5% for the seronegative group and 5.4% for the seropositive group. Genotype distributions were consistent with Hardy-Weinberg expectations in both groups, suggesting that none of the three mutations has a detectable selective effect. Difference in the allelic and genotypic frequencies was statistically significant for the CCR2 locus, the frequency in the seronegative group being twice that found in the seropositive group. This finding may indicate a protective effect of the CCR2-64I mutation in relation to HIV transmission. However, considering that the CCR2-64I mutation has been more strongly associated with a decreased risk for progression for AIDS than to the resistance to the HIV infection, this could reflect an aspect of population structure or a Type I error.

Human immunodeficiency virus prevalence, incidence, and residual risk of transmission by transfusions at Retrovirus Epidemiology Donor Study-II blood centers in Brazil

BACKGROUND: In Brazil nationally representative donor data are limited on human immunodeficiency virus (HIV) prevalence, incidence, and residual transfusion risk. The objective of this study was to analyze HIV data obtained over 24 months by the Retrovirus Epidemiology Donor Study-II program in Brazil. STUDY DESIGN AND METHODS: Donations reactive to third-and fourth-generation immunoassays (IAs) were further confirmed by a less-sensitive (LS) IA algorithm and Western blot (WB). Incidence was calculated for first-time (FT) donors using the LS-EIA results and for repeat donors with a model developed to include all donors with a previous negative donation. Residual risk was projected by multiplying composite FT and repeat donor incidence rates by HIV marker-negative infectious window periods. RESULTS: HIV prevalence among FT donors was 92.2/ 105 donations. FT and repeat donor and composite incidences were 38.5 (95% confidence interval [CI], 25.651.4), 22.5 (95% CI, 17.6-28.0), and 27.5 (95% CI, 22.0-33.0) per 100,000 person-years, respectively. Male and community donors had higher prevalence and incidence rates than female and replacement donors. The estimated residual risk of HIV transfusion transmission was 11.3 per 106 donations (95% CI, 8.4-14.2), which could be reduced to 4.2 per 106 donations (95% CI, 3.2-5.2) by use of individual-donation nucleic acid testing (NAT). CONCLUSION: The incidence and residual transfusion risk of HIV infection are relatively high in Brazil. Implementation of NAT will not be sufficient to decrease transmission rates to levels seen in the United States or Europe; therefore, other measures focused on decreasing donations by at-risk individuals are also necessary.

Quality of sleep and quality of life in adolescents infected with human immunodeficiency virus

Objectives: To assess sleep characteristics of adolescents infected by HIV, and to ascertain whether psychosocial aspects are associated to the quality of sleep. Methods: A cross-sectional study assessing 102 HIV-infected adolescents of both genders, aged between 10 and 20 years-old and 120 Controls. Data collection was performed by applying the Sleep Disturbance Scale for Children, the Epworth Sleepiness Scale, and the Pediatric Quality of Life Inventory. Results: A sleep disturbance prevalence of 77.4% was found in patients, and a 75% prevalence in controls, and there was correlation between quality of sleep and of life. HIV-infected adolescents scored higher for sleep breathing disorders and had higher prevalence of excessive daytime sleepiness. Conclusions: HIV-infected adolescents had similar quality of sleep compared to healthy adolescents. This may be explained by the steady improvements in daily living as a result of successful anti-retroviral therapy, and by the vulnerability that affects Brazilian adolescents living in major urban centers.

Distribution of human immunodeficiency virus type 1 subtypes in the State of Amazonas, Brazil, and subtype C identification

Few studies have reported the molecular epidemiological characterization of HIV-1 in the Northern region of Brazil. The present study reports the molecular and epidemiological characterization of 31 HIV-1 isolates from blood donors from the State of Amazonas who donated blood between April 2006 and March 2007. Serum/plasma samples from all donors were screened for HIV antibodies by ELISA and the results confirmed by Western blot analysis. Genomic DNA was extracted from the buffy coat using the Super Quik-Gene-DNA Isolation kit. Nested PCR was performed on the env, gag, and pol regions of HIV-1 using the Gene Amp PCR System 9700. Sequencing reactions were performed using the inner PCR primers and the DYEnamic (TM) ET Dye Terminator Kit, and phylogenetic analysis was performed using the gag, pol, and env gene sequences. We collected samples from 31 blood donors who tested positive for HIV-1 in confirmatory experiments. The male: female ratio of blood donors was 3.4:1, and the mean age was 32.4 years (range: 19 to 61 years). Phylogenetic analysis showed that subtype B is the most prevalent among Northern Brazilian HIV-1-seropositive blood donors. One HIV-1 subtype C and one circulating recombinant form (CRF_BF) of HIV-1 were identified in the State of Amazonas. This is the first study showing the occurrence of a possible "homogenous" subtype C in this region of Brazil. This finding could contribute to a better characterization of the HIV-1 strains that circulate in the country.

Correlates of human immunodeficiency virus cervicovaginal shedding among postmenopausal and fertile-aged women

Objective: The aims of this study were to compare the intensity of human immunodeficiency virus (HIV)-RNA genital shedding among postmenopausal (PM) and fertile-aged (F) women and to investigate the association between viral shedding and gynecological features, HIV plasma viral loads, and other markers of HIV disease progression. Methods: We interviewed 146 HIV-infected women (73 PM/73 F) in search of gynecological complaints and signs and symptoms of HIV disease and obtained additional information concerning HIV infection by medical chart review. Cervicovaginal lavages (CVLs) were collected for assessment of HIV shedding. Laboratory analyses included CD4(+) cell counts, HIV-RNA quantitation in plasma and CVL, and screening for concurrent genital infections. Results: HIV-RNA genital shedding was detected in 16.4% of PM and 21.9% of F women (P = 0.400), and the intensity of HIV shedding did not differ between both groups (means-PM: 1.4log/mL; F: 1.4log/mL; P = 0.587). Three women (2 PM/1 F) exhibited viral shedding in the absence of detectable viremia. HIV plasma viral loads correlated with HIV shedding in both groups. In multivariable analysis, HIV plasma viral loads were independently associated with HIV shedding in both groups. Moreover, the intensity of shedding was independently associated with vaginal pH, tumor necrosis factor a concentrations in CVL, and HIV plasma viral loads. Conclusions: Despite significant changes that occur in the vaginal mucosa of PM women, HIV cervicovaginal shedding was not significantly influenced by this state in our cohort. In contrast, increased vaginal pH and genital inflammation, evidenced by increased tumor necrosis factor alpha concentrations in CVL and HIV plasma viral loads, were independently associated with the intensity of HIV shedding in PM and F women.

Evaluation of rapid tests for human immunodeficiency virus as a tool to detect recent seroconversion

The identification of recent HIV infection is important for epidemiological studies and to monitor the epidemic. The objective of this study was to evaluate two rapid tests that are easily available to the Brazilian scientific community for using as markers of recent HIV infection. The Rapid Test - HIV-1/2 Bio-Manguinhos (Bio-Manguinhos/Fiocruz, Brazil) and the Rapid Check HIV 1&2 (NDI-UFES, Center for Infectious Diseases, Universidade Federal do Espirito Santo) were tested, using 489 samples with HIV positive serology, from blood donors, previously classified as recent or long-term infection by serological testing algorithm for recent HIV seroconversion (STARHS) or LS-HIV Vitros assay methods. The samples were diluted prior to testing (1:50 and 1:100 for the Rapid Test - HIV-1/2 Bio-Manguinhos, and 1:500 and 1:600 for the Rapid Check HIV 1&2). Negative samples were considered recent infection, whereas those showing any color intensity were associated with long-term infection. The best dilutions were 1:100 for HIV-1/2 Bio-Manguinhos test (Kappa = 0.840; overall agreement = 0.93), and 1:500 for the Rapid Check HIV 1&2 (Kappa = 0.867; overall agreement = 0.94). The results suggest that both rapid tests can be used to detect recent seroconversion. (C) 2012 Elsevier Editora Ltda. All rights reserved.

Distribution of human immunodeficiency virus type 1 subtypes in the state of Amazonas, Brazil, and subtype C identification

Few studies have reported the molecular epidemiological characterization of HIV-1 in the Northern region of Brazil. The present study reports the molecular and epidemiological characterization of 31 HIV-1 isolates from blood donors from the State of Amazonas who donated blood between April 2006 and March 2007. Serum/plasma samples from all donors were screened for HIV antibodies by ELISA and the results confirmed by Western blot analysis. Genomic DNA was extracted from the buffy coat using the Super Quik-Gene-DNA Isolation kit. Nested PCR was performed on the env, gag, and pol regions of HIV-1 using the Gene Amp PCR System 9700. Sequencing reactions were performed using the inner PCR primers and the DYEnamic™ ET Dye Terminator Kit, and phylogenetic analysis was performed using the gag, pol, and env gene sequences. We collected samples from 31 blood donors who tested positive for HIV-1 in confirmatory experiments. The male:female ratio of blood donors was 3.4:1, and the mean age was 32.4 years (range: 19 to 61 years). Phylogenetic analysis showed that subtype B is the most prevalent among Northern Brazilian HIV-1-seropositive blood donors. One HIV-1 subtype C and one circulating recombinant form (CRF_BF) of HIV-1 were identified in the State of Amazonas. This is the first study showing the occurrence of a possible "homogenous" subtype C in this region of Brazil. This finding could contribute to a better characterization of the HIV-1 strains that circulate in the country.

Evaluation of glycoconjugate antigens as vaccine candidates against group A Streptococcus and human immunodeficiency virus infections

This PhD thesis discusses the rationale for design and use of synthetic oligosaccharides for the development of glycoconjugate vaccines and the role of physicochemical methods in the characterization of these vaccines. The study concerns two infectious diseases that represent a serious problem for the national healthcare programs: human immunodeficiency virus (HIV) and Group A Streptococcus (GAS) infections. Both pathogens possess distinctive carbohydrate structures that have been described as suitable targets for the vaccine design. The Group A Streptococcus cell membrane polysaccharide (GAS-PS) is an attractive vaccine antigen candidate based on its conserved, constant expression pattern and the ability to confer immunoprotection in a relevant mouse model. Analysis of the immunogenic response within at-risk populations suggests an inverse correlation between high anti-GAS-PS antibody titres and GAS infection cases. Recent studies show that a chemically synthesized core polysaccharide-based antigen may represent an antigenic structural determinant of the large polysaccharide. Based on GAS-PS structural analysis, the study evaluates the potential to exploit a synthetic design approach to GAS vaccine development and compares the efficiency of synthetic antigens with the long isolated GAS polysaccharide. Synthetic GAS-PS structural analogues were specifically designed and generated to explore the impact of antigen length and terminal residue composition. For the HIV-1 glycoantigens, the dense glycan shield on the surface of the envelope protein gp120 was chosen as a target. This shield masks conserved protein epitopes and facilitates virus spread via binding to glycan receptors on susceptible host cells. The broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose oligosaccharides on the gp120 subunit of HIV-1 Env protein. This oligomannose epitope has been a subject to the synthetic vaccine development. The cluster nature of the 2G12 epitope suggested that multivalent antigen presentation was important to develop a carbohydrate based vaccine candidate. I describe the development of neoglycoconjugates displaying clustered HIV-1 related oligomannose carbohydrates and their immunogenic properties.

Human immunodeficiency virus type 1 and influenza virus exit via different membrane microdomains

Directed release of human immunodeficiency virus type 1 (HIV-1) into the cleft of the virological synapse that can form between infected and uninfected T cells, for example, in lymph nodes, is thought to contribute to the systemic spread of this virus. In contrast, influenza virus, which causes local infections, is shed into the airways of the respiratory tract from free surfaces of epithelial cells. We now demonstrate that such differential release of HIV-1 and influenza virus is paralleled, at the subcellular level, by viral assembly at different microsegments of the plasma membrane of HeLa cells. HIV-1, but not influenza virus, buds through microdomains containing the tetraspanins CD9 and CD63. Consequently, the anti-CD9 antibody K41, which redistributes its antigen and also other tetraspanins to cell-cell adhesion sites, interferes with HIV-1 but not with influenza virus release. Altogether, these data strongly suggest that the bimodal egress of these two pathogenic viruses, like their entry into target cells, is guided by specific sets of host cell proteins.

JC virus-specific immune responses in human immunodeficiency virus type 1 patients with progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a frequently fatal disease caused by uncontrolled polyomavirus JC (JCV) in severely immunodeficient patients. We investigated the JCV-specific cellular and humoral immunity in the Swiss HIV Cohort Study. We identified PML cases (n = 29), as well as three matched controls per case (n = 87), with prospectively cryopreserved peripheral blood mononuclear cells and plasma at diagnosis. Nested controls were matched according to age, gender, CD4(+) T-cell count, and decline. Survivors (n = 18) were defined as being alive for >1 year after diagnosis. Using gamma interferon enzyme-linked immunospot assays, we found that JCV-specific T-cell responses were lower in nonsurvivors than in their matched controls (P = 0.08), which was highly significant for laboratory- and histologically confirmed PML cases (P = 0.004). No difference was found between PML survivors and controls or for cytomegalovirus-specific T-cell responses. PML survivors showed significant increases in JCV-specific T cells (P = 0.04) and immunoglobulin G (IgG) responses (P = 0.005). IgG responses in survivors were positively correlated with CD4(+) T-cell counts (P = 0.049) and negatively with human immunodeficiency virus RNA loads (P = 0.03). We conclude that PML nonsurvivors had selectively impaired JCV-specific T-cell responses compared to CD4(+) T-cell-matched controls and failed to mount JCV-specific antibody responses. JCV-specific T-cell and IgG responses may serve as prognostic markers for patients at risk.

Distribution of human immunodeficiency virus (HIV) in the CNS of children with severe HIV encephalomyelopathy

The presence and distribution of human immunodeficiency virus (HIV) were examined in the CNS of two children with severe HIV encephalitis and myelitis. Using polymerase chain reaction-mediated DNA amplification and subsequent Southern analysis, proviral HIV gag sequences were identified in brain tissue of both patients. In situ hybridization using antisense oligonucleotide probes revealed abundant HIV gag and env/nef RNAs selectively in areas with histopathological evidence for HIV-induced tissue damage. The spinal cord of one patient exhibited a striking subpial accumulation of HIV RNAs strongly suggestive of a liquorigenic spread of the infection. HIV RNAs were typically associated with cells of the monocyte/macrophage lineage, as shown by a combined immunohistochemical and in situ hybridization procedure. The present study supports the view that the pattern and distribution of HIV-induced brain lesions is largely determined by the extent of focal HIV replication within the CNS.