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.

Treatment modification in human immunodeficiency virus-infected individuals starting combination antiretroviral therapy between 2005 and 2008

Adverse effects of combination antiretroviral therapy (CART) commonly result in treatment modification and poor adherence.

Determinants of hepatitis A vaccine immunity in a cohort of human immunodeficiency virus-infected children living in Switzerland

Vaccination in HIV-infected children is often less effective than in healthy children. The goal of this study was to assess vaccine responses to hepatitis A virus (HAV) in HIV-infected children. Children of the Swiss Mother and Child HIV Cohort Study (MoCHiV) were enrolled prospectively. Recommendations for initial, catch-up, and additional HAV immunizations were based upon baseline antibody concentrations and vaccine history. HAV IgG was assessed by enzyme-linked immunosorbent assay (ELISA) with a protective cutoff value defined as ≥10 mIU/ml. Eighty-seven patients were included (median age, 11 years; range, 3.4 to 21.2 years). Forty-two patients were seropositive (48.3%) for HAV. Among 45 (51.7%) seronegative patients, 36 had not received any HAV vaccine dose and were considered naïve. Vaccine responses were assessed after the first dose in 29/35 naïve patients and after the second dose in 33/39 children (25 initially naïve patients, 4 seronegative patients, and 4 seropositive patients that had already received 1 dose of vaccine). Seroconversion was 86% after 1 dose and 97% after 2 doses, with a geometric mean concentration of 962 mIU/ml after the second dose. A baseline CD4(+) T cell count below 750 cells/μl significantly reduced the post-2nd-dose response (P = 0.005). Despite a high rate of seroconversion, patients with CD4(+) T cell counts of <750/μl had lower anti-HAV antibody concentrations. This may translate into a shorter protection time. Hence, monitoring humoral immunity may be necessary to provide supplementary doses as needed.

Orosomucoid (alpha1-acid glycoprotein) plasma concentration and genetic variants: effects on human immunodeficiency virus protease inhibitor clearance and cellular accumulation

BACKGROUND AND OBJECTIVE: Protease inhibitors are highly bound to orosomucoid (ORM) (alpha1-acid glycoprotein), an acute-phase plasma protein encoded by 2 polymorphic genes, which may modulate their disposition. Our objective was to determine the influence of ORM concentration and phenotype on indinavir, lopinavir, and nelfinavir apparent clearance (CL(app)) and cellular accumulation. Efavirenz, mainly bound to albumin, was included as a control drug. METHODS: Plasma and cells samples were collected from 434 human immunodeficiency virus-infected patients. Total plasma and cellular drug concentrations and ORM concentrations and phenotypes were determined. RESULTS: Indinavir CL(app) was strongly influenced by ORM concentration (n = 36) (r2 = 0.47 [P = .00004]), particularly in the presence of ritonavir (r2 = 0.54 [P = .004]). Lopinavir CL(app) was weakly influenced by ORM concentration (n = 81) (r2 = 0.18 [P = .0001]). For both drugs, the ORM1 S variant concentration mainly explained this influence (r2 = 0.55 [P = .00004] and r2 = 0.23 [P = .0002], respectively). Indinavir CL(app) was significantly higher in F1F1 individuals than in F1S and SS patients (41.3, 23.4, and 10.3 L/h [P = .0004] without ritonavir and 21.1, 13.2, and 10.1 L/h [P = .05] with ritonavir, respectively). Lopinavir cellular exposure was not influenced by ORM abundance and phenotype. Finally, ORM concentration or phenotype did not influence nelfinavir (n = 153) or efavirenz (n = 198) pharmacokinetics. CONCLUSION: ORM concentration and phenotype modulate indinavir pharmacokinetics and, to a lesser extent, lopinavir pharmacokinetics but without influencing their cellular exposure. This confounding influence of ORM should be taken into account for appropriate interpretation of therapeutic drug monitoring results. Further studies are needed to investigate whether the measure of unbound drug plasma concentration gives more meaningful information than total drug concentration for indinavir and lopinavir.

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.

A randomized clinical trial of mupirocin in the eradication of Staphylococcus aureus nasal carriage in human immunodeficiency virus disease

Seventy-six human immunodeficiency virus (HIV)-infected patients with Staphylococcus aureus nasal carriage were randomized to treatment groups receiving intranasal mupirocin or placebo twice daily for 5 days. Nasal cultures for S. aureus were obtained at 1, 2, 6, and 10 weeks after therapy. At 1 week, 88% of mupirocin-treated patients had negative nasal cultures compared with 8% in placebo patients (P<.001). The percentage of mupirocin-treated patients with persistently negative nasal cultures decreased over time (63%, 45%, and 29% at 2, 6, and 10 weeks, respectively) but remained significantly greater than the placebo group (3% at 2, 6, and 10 weeks). In mupirocin-treated patients, most (16/19) instances of nasal recolonization were with pretreatment strains (determined by means of by pulsed field gel electrophoresis); mupirocin resistance was not observed. Five days of treatment with mupirocin eliminated S. aureus nasal carriage in HIV-infected patients for several weeks; however, since the effect waned over time, intermittent dosing regimens should be considered for long-term eradication.

Outcome of treated anogenital intraepithelial neoplasia among human immunodeficiency virus-infected women

OBJECTIVE: To determine characteristics and clinical course of high-grade anogenital intraepithelial neoplasia (AIN) in human immunodeficiency virus (HIV)-infected women. STUDY DESIGN: HIV-positive women with biopsy-proven high-grade (II and III) vulvar (VIN), vaginal (VAIN) or perianal intraepithelial neoplasia (PAIN) were identified in the electronic databases of 2 colposcopy clinics. RESULTS: A total of 31 patients were identified from 1992 to 2007, of which 30 had a mean follow-up of 47.7 months (SD = 46.0; range, 2.6-166.2). Of the patients, 77.4% had VIN, 12.9% VAIN and 9.7% PAIN at first diagnosis. Age at diagnosis of IN was 36.2 years (SD +/- 5.2; range, 23.5-47.0). Ninety percent of patients received antiretroviral therapy at first diagnosis of IN; 65% (13 of 20) of patients with a follow-up of > 2 years required a second treatment, and 2 developed invasive vulvar cancer (10%). CONCLUSION: AIN among HIV-positive women shows a high relapse rate despite treatment modality used and a substantial invasive potential.

Determinants of vaccine immunity in the cohort of human immunodeficiency virus-infected children living in Switzerland

BACKGROUND: Human immunodeficiency virus (HIV)-infected children are at increased risk of infections caused by vaccine preventable pathogens, and specific immunization recommendations have been issued. METHODS: A prospective national multicenter study assessed how these recommendations are followed in Switzerland and how immunization history correlates with vaccine immunity. RESULTS: Among 87 HIV-infected children (mean age: 11.1 years) followed in the 5 Swiss university hospitals and 1 regional hospital, most (76%) had CD4 T cells >25%, were receiving highly active antiretroviral treatment (79%) and had undetectable viral load (60%). Immunization coverage was lower than in the general population and many lacked serum antibodies to vaccine-preventable pathogens, including measles (54%), varicella (39%), and hepatitis B (65%). The presence of vaccine antibodies correlated most significantly with having an up-to-date immunization history (P<0.05). An up-to-date immunization history was not related to age, immunologic stage, or viremia but to the referral medical center. CONCLUSIONS: All pediatricians in charge of HIV-infected children are urged to identify missing immunizations in this high-risk population.

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.

Isolation of Lautropia mirabilis from oral cavities of human immunodeficiency virus-infected children.

Lautropia mirabilis, a pleomorphic, motile, gram-negative coccus, has been isolated from the oral cavities of 32 of 60 (53.3%) children infected with human immunodeficiency virus (HIV) and 3 of 25 (12.0%) HIV-uninfected controls; the association of L. mirabilis isolation with HIV infection is significant (P < 0.001). All children in the study, both HIV-infected children and controls, were born to HIV-infected mothers. The presence of this bacterium was not associated with clinical disease in these children. The HIV-infected children with L. mirabilis did not differ from the HIV-infected children without L. mirabilis in immunological status, clinical status, or systemic medications. The role of HIV infection itself or concomitant factors in the establishment of L. mirabilis in the oral cavity remains to be elucidated.

{\it In vivo\/} induction of cytotoxic T lymphocytes against human immunodeficiency virus type 1 by synthetic viral peptides

Cytotoxic T lymphocytes (CTLs) play an important role in the suppression of initial viremia after acute infection with the human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS). Most HIV-infected individuals attain a high titer of anti-HIV antibodies within weeks of infection; however this antibody-mediated immune response appears not to be protective. In addition, anti-HIV antibodies can be detrimental to the immune response to HIV through enhancement of infection and participating in autoimmune reactions as a result of HIV protein mimicry of self antigens. Thus induction and maintenance of a strong HIV-specific CTL immune response in the absence of anti-HIV antibodies has been proposed to be the most effective means of controlling of HIV infection. Immunization with synthetic peptides representing HIV-specific CTL epitopes provides a way to induce specific CTL responses, while avoiding stimulation of anti-HIV antibody. This dissertation examines the capacity of synthetic peptides from the V3 loop region of the gp120 envelope protein from several different strain of HIV-1 to induce HIV-specific, MHC-restricted CD8$\sp+$ CTL response in vivo in a mouse model. Seven synthetic peptides representative of sequences found throughout North America, Europe, and Central Africa have been shown to prime CTLs in vivo. In the case of the MN strain of HIV-1, a 13 amino acid sequence defining the epitope is most efficient for optimal induction of specific CTL, whereas eight to nine amino acid sequences that could define the epitope were not immunogenic. In addition, synthesis of peptides with specific amino acid substitutions that are important for either MHC binding or T cell receptor recognition resulted in peptides that exhibited increased immunogenicity and induced CTLs that displayed altered specificity. V3 loop peptides from HIV-1 MN, SC, and Z321 induced a CTL population that was broadly cross-reactive against strains of HIV-1 found throughout the world. This research confirms the potential efficacy of using synthetic peptides for in vivo immunization to induce HIV-specific CTL-mediated responses and provides a basis for further research into development of synthetic peptide-based vaccines. ^

The role of substance abuse treatment in preventing human immunodeficiency virus transmission among injecting drug users

This exploratory study assesses the utility of substance abuse treatment as a strategy for preventing human immunodeficiency virus (HIV) transmission among injecting drug users (IDUs). Data analyzed in this study were collected in San Antonio, TX, 1989 through 1995 using both qualitative and quantitative methods. Qualitative data included ethnographic interviews with 234 active IDUs; quantitative data included baseline risk assessments and HIV screening plus interviews follow-up interviews administered approximately six months later to 823 IDUs participating in a Federally-funded AIDS community outreach demonstration project.^ Findings that have particularly important implications for substance abuse treatment as an HIV prevention strategy for IDUs are listed below. (1) IDUs who wanted treatment were significantly more likely to be daily heroin users. (2) IDUs who want treatment were significantly more likely to have been to treatment previously. (3) IDUs who wanted treatment at baseline reported significantly higher levels of HIV risk than IDUs who did not want treatment. (4) IDUs who went to treatment between their baseline and follow-up interviews reported significantly higher levels of HIV risk at baseline than IDUs who did not go to treatment. (5) IDUs who went to treatment between their baseline and follow-up interviews reported significantly greater decreases in injection-related HIV risk behaviors. (6) IDUs who went to treatment reported significantly greater decreases in sexual HIV risk behaviors than IDUs who did not go to treatment.^ This study also noted a number of factors that may limit the effectiveness of substance abuse treatment in reducing HIV risk among IDUs. Findings suggest that the impact of methadone maintenance on HIV risk behaviors among opioid dependent IDUs may be limited by the negative manner in which it is perceived by IDUs as well as other elements of society. One consequence of the negative perception of methadone maintenance held by many elements of society may be an unwillingness to provide public funding for an adequate number of methadone maintenance slots. Thus many IDUs who would be willing to enter methadone maintenance are unable to enter it and many IDUs who do enter it are forced to drop out prematurely. ^