HIV-1 Sera Research Articles

Antibody response to mycobacterial Rpf B protein and its immunodominant peptides in HIV-TB co-infected individuals

Diagnosis of TB at early stages of HIV infection may lead to timely intervention for improving patient outcome. Antibodies to Mycobacterium tuberculosis recombinant RpfB protein and two immunodominant peptides of Rpf B protein were evaluated in the sera of HIV +TB+, HIV+ and HIV− pulmonary TB patients by ELISA. Serum antibodies from 90 % and 65 % of HIV+TB+ patients reacted to recombinant RpfB protein and synthetic peptide RpfP1 respectively. Overall, this study shows that resuscitation promoting factor B elicits humoral antibody response in HIV+TB+ co-infected individuals and be proposed as a potential biomarker for diagnosis of HIV+TB+ patients, however further longitudinal follow up studies are warranted.

Metabolite variations in the sera of HIV+ patients after an oral administration of effervescent glutamine and in comparison to non-HIV individuals by NMR.

It was demonstrated that effervescent glutamine supplementation in HIV+ individuals treated with antiretroviral therapy (ART) increased CD4+ T lymphocytes, decreased inflammation biomarkers, and brought health benefits. This pilot study aimed to explore serum metabolite variations in the HIV+ group under ART after 30 days of supplementation with glutamine, and in comparison to the matched HIV- group. The group of HIV+ showed lower levels of choline, creatine, pyruvate, glutamate, lysine, and tyrosine when compared to the HIV- group. Glucose, lipids, lactate, glutamine, phenylalanine, threonine, and phenylalanine/tyrosine were higher in HIV+ patients under long ART. Serum metabolome variations were shown to be consistent with the health improvements observed in the HIV+ group after effervescent glutamine supplementation, which might aid in ART in HIV+ individuals.

Novel HIV-1 miRNAs stimulate TNFα release in human macrophages via TLR8 signaling pathway.

PurposeTo determine whether HIV-1 produces microRNAs and elucidate whether these miRNAs can induce inflammatory response in macrophages (independent of the conventional miRNA function in RNA interference) leading to chronic immune activation.MethodsUsing sensitive quantitative Real Time RT-PCR and sequencing, we detected novel HIV-derived miRNAs in the sera of HIV+ persons, and associated with exosomes. Release of TNFα by macrophages challenged with HIV miRNAs was measured by ELISA.ResultsHIV infection of primary alveolar macrophages produced elevated levels of viral microRNAs vmiR88, vmiR99 and vmiR-TAR in cell extracts and in exosome preparations from conditioned medium. Furthermore, these miRNAs were also detected in exosome fraction of sera from HIV-infected persons. Importantly, vmiR88 and vmiR99 (but not vmiR-TAR) stimulated human macrophage TNFα release, which is dependent on macrophage TLR8 expression. These data support a potential role for HIV-derived vmiRNAs released from infected macrophages as contributing to chronic immune activation in HIV-infected persons, and may represent a novel therapeutic target to limit AIDS pathogenesis.ConclusionNovel HIV vmiR88 and vmiR99 are present in the systemic circulation of HIV+ persons and could exhibit biological function (independent of gene silencing) as ligands for TLR8 signaling that promote macrophage TNFα release, and may contribute to chronic immune activation. Targeting novel HIV-derived miRNAs may represent a therapeutic strategy to limit chronic immune activation and AIDS progression.

Characteristics and Outcome of Minority Group Patients in Methadone Maintenance Treatment

Minority status is associated with mental and physical morbidity, substance dependence, and poor outcomes. To compare characteristics and treatment outcomes between patients from two minority groups in Israel (Christians and Muslims) and patients from the majority population (Jews) in methadone maintenance treatment (MMT), we prospectively studied all patients admitted to our clinic between 1993 and 2012 and followed up until 2013; 655 Jews, 67 Christians, and 37 Muslims. Christian patients differed from Jews and Muslims by younger age at admission to MMT, greater prevalence of drug injectors, and a higher proportion of Hepatitis-C and HIV sera positive. Muslims had comparatively less education and a lower proportion of females. The three groups had similar rates of one-year retention (75.9%) and opiate abstinence (68.1%). They also did not differ in long-term retention (up to 20 years): Muslims 5.5 years (95%CI 3.6-7.4), Christians 7.5 years (95%CI 6-9.1), and Jews 7.6 years (95%CI 7-8.2, p = .3). The Hepatitis-C incidence, however, was higher among the 21 admitted Hepatitis-C seronegative minorities (5.0/100 person years) than the 207 Hepatitis-C seronegative non-minority patients (1.7/100 person years, p=0.03). All groups had good treatment outcomes, except for Hepatitis-C seroconversion, which necessitates a specific preventive intervention among the minority groups.

Methadone Maintenance Treatment Experience in Macao – Prospective Follow-up for Initial 4.5 Years

The initiation of the first methadone maintenance treatment program (MMT) in Macao was founded in collaboration between MMT clinics in the USA and Israel. All patients admitted into treatment between October 2005 and October 2008 were prospectively followed through March 2010. Of the 163 patients, 81% were male, the mean age on admission was 39.5 (sd = 10.2). Seventy-three percent (n = 119) were hepatitis C sera positive, and 4.9% (n = 8) were HIV sera positive. One-year treatment retention rate was 59.5%, with 52.6% of the 95 patients who stayed in treatment having an opiate-negative urine test at the 10-month evaluation. Four and a half years of follow-up showed mean long-term retention (Kaplan Meier analyses) of 2.2 years. Higher methadone dose (≥80mg/day) and hepatitis C sera positive status were predictors for longer treatment retention. This study describes an effective model of MMT that supports the expansion of addiction treatment in other countries.

HIV-1 tat promotes integrin-mediated HIV transmission to dendritic cells by binding Env spikes and competes neutralization by anti-HIV antibodies.

Use of Env in HIV vaccine development has been disappointing. Here we show that, in the presence of a biologically active Tat subunit vaccine, a trimeric Env protein prevents in monkeys virus spread from the portal of entry to regional lymph nodes. This appears to be due to specific interactions between Tat and Env spikes that form a novel virus entry complex favoring R5 or X4 virus entry and productive infection of dendritic cells (DCs) via an integrin-mediated pathway. These Tat effects do not require Tat-transactivation activity and are blocked by anti-integrin antibodies (Abs). Productive DC infection promoted by Tat is associated with a highly efficient virus transmission to T cells. In the Tat/Env complex the cysteine-rich region of Tat engages the Env V3 loop, whereas the Tat RGD sequence remains free and directs the virus to integrins present on DCs. V2 loop deletion, which unshields the CCR5 binding region of Env, increases Tat/Env complex stability. Of note, binding of Tat to Env abolishes neutralization of Env entry or infection of DCs by anti-HIV sera lacking anti-Tat Abs, which are seldom present in natural infection. This is reversed, and neutralization further enhanced, by HIV sera containing anti-Tat Abs such as those from asymptomatic or Tat-vaccinated patients, or by sera from the Tat/Env vaccinated monkeys. Thus, both anti-Tat and anti-Env Abs are required for efficient HIV neutralization. These data suggest that the Tat/Env interaction increases HIV acquisition and spreading, as a mechanism evolved by the virus to escape anti-Env neutralizing Abs. This may explain the low effectiveness of Env-based vaccines, which are also unlikely to elicit Abs against new Env epitopes exposed by the Tat/Env interaction. As Tat also binds Envs from different clades, new vaccine strategies should exploit the Tat/Env interaction for both preventative and therapeutic interventions.

Hepatitis Be Antigen (HBeAg) and Antibody (HBeAb) in Human Immunodeficiency Virus (HIV) Seropositive Patients in Lagos, Nigeria.

This preliminary study investigated the incidence of HBeAg and anti-HBe antibodies in the sera of HIV patients in Lagos, Nigeria, during the month of June, 2005. A total of 96 patients, aged 5-65 years who had positive HIV sera comprised the study population. The confirmation test was done at NIMR, Lagos. HIV sera were characterized by Enzyme Linked Immuno-sorbent Assay (ELISA) for HBeAg and HBeAb. Hepatitis Be antigen were present in the sera of seropositive HIV patients. In the studied group the incidence rate is low with the presence of antibodies in some cases. It is difficult to categorize those with antibodies as immune because their surface antigens were not known. Three samples were positive for the antigen, a male in the age group 11-20 and two females in the group 21-30. The presence of antibodies cut across the various age groups: 1 in 1-10, 2 in 21-30 group with each sex having one each; five females and two males in age groups 31-40; one male in age group 41-50; 1 female in age group 51-60. Age groups 1-10 and 61-70 have no positive antibodies in their sera. It is suggested that this preliminary study is practiable, but that it is only a pointer, and a rough guide to the incidence in Lagos.

Development and studies of the anti-R7V neutralizing antibody ELISA test: A new serological test for HIV seropositive patients

A new peptide-based ELISA test developed for the detection of anti-R7V specific antibodies in the sera of HIV seropositive patients is described. HIV virus acquires a cellular antigen at the moment the virus is released by budding, the R7V epitope. Certain patients produce anti-R7V Ab which are described as having the capacity to neutralize in vitro cell infection by HIV. Anti-R7V Ab are also detected in asymptomatic patients who have a lower likelihood of progressing to AIDS. Tested with 449 serum samples, the prevalence of anti-R7V Ab was 53.2% in HIV positive patients and 5.5% in HIV negative subjects. According to the duration of infection, the seroprevalence reaches almost 80% of non-treated long-term infected patients. Other retrospective studies were conducted on 308 HIV positive samples; the presence of anti-R7V Ab was significantly higher for 177 asymptomatic patients (64.4%) compared to the 131 symptomatic patients (35.1%). Regarding their neutralizing ability, anti-R7V antibodies were detected at the highest percentage in asymptomatic HIV-infected patients naive of treatment. Besides conventional biological parameters (CD4 and viral load), the detection of anti-R7V antibodies could be proposed to clinicians as an additional tool to manage treatment initiation and to improve the psychological state of their asymptomatic patients.

Dichotomy in cross‐clade reactivity and neutralization by HIV‐1 sera: Implications for active and passive immunotherapy

The identification of broadly reactive and cross-clade neutralizing antibodies will facilitate the development of a more universally effective vaccine for human immunodeficiency virus (HIV). Antibodies in sera from individuals infected with Clade B HIV bind native primary viral isolates, and virus binding correlates with neutralization and stable clinical disease. In this study, we quantified cross-clade antibody reactivity and neutralization by Clades B and C sera. Primary viral isolates were captured by serum IgG bound to anti-human IgG and quantitated as p24 released by lysis of captured virus. Neutralization was determined using PHA-stimulated PBMC. Clade B antibodies reacted more frequently with Clade B R5 virus, but positive sera captured quantitatively more X4 virus than R5 and R5X4 virus. Clade B sera reacted less frequently and captured less Clade C virus than Clade B virus. Antibodies in Clade C sera captured Clades B and C isolates with equal frequency and quantity. There was no difference in neutralization of Clade B virus by either group of sera; however, Clade C sera neutralized Clade C virus, whereas Clade B sera were ineffective against Clade C virus. Thus, there are distinct differences in cross-clade reactivity of and neutralization by antibodies induced in response to Clade C infection compared to Clade B infection. Understanding antibody responses to native virions after Clade C infection and cross clade antibody behavior has implications for understanding pathogenesis and vaccine development.

Immunolocalization Studies of an Antisense Protein in HIV-1-Infected Cells and Viral Particles

The minus DNA strand of HIV-1 presents an open reading frame that is complementary to the HIV-1 envelope messenger, is highly conserved among HIV-1 isolates, and may encode a hydrophobic protein. In previous studies, the antisense transcript has been identified both in various HIV-infected cell lines and in leukocytes of HIV-1+ patients. The expression of the corresponding antisense protein (ASP) during natural HIV-1 infection has been indirectly evidenced by the identification of anti-ASP antibodies in the sera of HIV+ patients. We have used immunoelectron microscopy procedures (ultrasmall gold particles coupled to silver enhancement) to establish direct evidence of ASP production. ASP has then been detected in chronically and acutely HIV-1-infected cell lines. The protein, found mostly associated with various cellular membranes as well as with the virions released from the cells, indicated that ASP is a bona fide component for the virions and may participate in the particle formation.

Similar HIV-1 subtype A V3 loop sequences are found in dual HIV-1/HIV-2 and HIV-1-only seropositives in Ghana.

775 G H AN A A N D IV ORY COAS T are situated between the two major initial foci of the African HIV-1 and HIV-2 epidemics. It is in such countries that dual HIV-1/HIV-2 serological reactivity (dual seropositivity) is most common. The proportion of dual seropositivity has increased in West Africa countries, where HIV-2 was initially the main cause of the epidemic. The diagnosis of dual HIV-1/HIV-2 infections may be complicated, as HIV-2 DNA cannot always be detected and the levels of HIV-2 DNA in dual seropositives may decrease with progression to AIDS. Also, even low levels of HIV-2 antibodies can imply dual infection. 4 The lack of HIV-2 DNA and/or low HIV2 antibody level may thus be related not only to the diagnostic methods used, but possibly to suppression of HIV-2 by HIV-1. HIV-2 infection may protect against HIV-1 superinfection, which could have impact on the development of protective vaccines. It has been suggested that the HIV-2 protection from subsequent HIV-1 infection is mediated by b -chemokines and CD8 1 cells, and is restricted to HIV-1 strains using the CCR5 receptor. Furthermore, conflicting reports on cross-neutralization of HIV-1 isolates by HIV-2 sera exist. Since the V3 loops of the viruses are important for chemokine receptor usage and neutralization, a critical look at this region is of interest for understanding the interaction between HIV-1 and HIV-2 in vivo. We therefore determined the V3 sequences of HIV-1 strains obtained from epidemiologicall y matched patients seropositive for both types or HIV-1 only. Peripheral blood mononuclear cells (PBMCs) were obtained from patients who were HIV seropositive by World Health Organization (WHO) criteria (Table 1), attending semi rural or urban AIDS clinics in the southern part of Ghana during 1996. Ethical permission was obtained from the Ministry of Health (Accra, Ghana), the University of Ghana Medical School (Accra, Ghana), and the Huddinge Hospital (Huddinge/ Stockholm, Sweden). Lymphocyte subset determination was performed by a flow cytometric method and the patients classfied as asymptomatic or symptomatic on the basis of the WHO expanded clinical definition of AIDS. Two commercial kits were used for analysis of anti-HIV reactivity. In brief, a dot immunobinding assay (Target HIV1-HIV2; V-Tech, Pomona, CA), which is based on homologous synthetic peptides in the HIV-1 and HIV2 transmembrane proteins, was used for screening. Confirmatory tests with a line immunoassay including recombinant proteins and peptides for HIV-1, and a peptide for HIV-2 (Innolia; NV Innogenetics, Antwerp, Belgium), were done according to the manufacturer instructions. Samples that reacted with HIV1 gp41 (and other bands) with a rating of 1 1 or above were considered HIV-1 seropositive, and those having additional reactivities of at least 11 with the HIV-2 gp36 band were classified as dual seropositive. Two million PBMCs were lysed and DNA was extracted by phenol±chloroform and ethanol precipitation. To amplify an env fragment encoding the V3 loop, the following outer primers were used: JA 167 [5 9 TAT C(C/T)T TTG AGC CAA TTC C(C/T)A TAC A] and JA 170 [5 9 GTG ATG TAT T(A/G)C A(A/G)T AGA AAA ATT C], with conditions previously described. For the nested step, primers V3-3 (59 biotin-AATGGCAGTCTAGCAGAA) and V3-4-M13 (5 9 M13-CTGGGTCCCCTCCTGAGG) were used. Direct sequencing was performed using magnetic beads with covalently coupled streptavidin (Dynabeads, M280-streptivid in; Dynal A.S., Oslo, Norway), an AutoRead sequencing kit (Pharmacia Biotech, Uppsala, Sweden), and an automated laser fluorescence sequencer (ALFexpress; Pharmacia Biotech). Sequences approximately 252 or 249 nucleotides long were manually aligned. The phylogenetic analysis was done with the TREEDON package and 1000 bootstrap resamplings. Distance calculations were derived by neighbor joining, using the Jin and Nei method ( g distribu-

Trans-Activation of the HIV type 1 promoter by 7,8-dihydroneopterin in vitro.

Infection with human immunodeficiency virus type 1 and 2 is associated with elevated concentrations of neopterin, released in large quantities by human macrophages on stimulation with interferon gamma. Evidence has suggested a potential role of neopterin derivatives in oxygen radical-mediated processes. Here we show that the redox-sensitive transcription factors AP-1 and NF-kappaB are activated by 7,8-dihydroneopterin, either directly (AP-1), or by the synergistic action with tumor necrosis factor alpha (NF-kappaB). We could further demonstrate that 7,8-dihydroneopterin enhances HIV-1 expression as shown in transient transfection assays using HIV-1 CAT promoter-reporter gene constructs. In sera of HIV+ patients 7,8-dihydroneopterin significantly correlated with neopterin and HIV-1 p24 antigen. On the basis of our data we therefore assume that 7,8-dihydroneopterin might augment progression to higher stages of HIV-associated disease.

The neutralization relationship of HIV type 1, HIV type 2, and SIVcpz is reflected in the genetic diversity that distinguishes them.

Neutralizing antibody (NA) patterns in the sera of individuals naturally infected with human immunodeficiency virus (HIV) type 1, HIV-2, and the simian immunodeficiency virus (SIVcpz) to their homologous and heterologous isolates were determined in a peripheral blood mononuclear cell-based neutralization assay. We examined the role of the V3 loop of HIV-1 and SIVcpz in neutralization and the cross-reactivities among them. Cross-neutralization by sera of humans and chimpanzees naturally infected, respectively, with HIV-1 and SIVcpz isolates was more extensive than the infrequent and low-titer cross-neutralizations observed between HIV-1 and HIV-2. Neutralization of 9 of the 16 HIV-1 isolates by 9 of 10 HIV-2 and all 3 SIVcpz antibody-positive sera were weak and sporadic (titer, 1:10-1:160). Twelve of 15 HIV-1 sera neutralized the 2 SIVcpz isolates with titers of 1:10-1:320 but only sporadically neutralized the 6 HIV-2 isolates (titers: 1:10-1:20). The majority of HIV-1 and SIVcpz sera bound to the V3 peptides although their binding capacity did not readily reflect their neutralizing capacity. The HIV-2 sera did not or only weakly bound to the V3 peptides. These results suggest that HIV-1 and SIVcpz share some structural and functional similarities that set them apart from HIV-2.

Cross-reactivity between autoimmune anti-U1 snRNP antibodies and neutralizing epitopes of HIV-1 gp120/41.

We report extensive amino acid sequence homology between HIV-1 gp120/41, and > 33% of a U1 RNA-associated splicing protein, 70K. The latter is a target of autoimmune anti-RNP antibodies in mixed connective tissue disease (MCTD). The homologies, involving dominant epitopes of 70K and neutralizing epitopes of gp120/41, are the basis for mutual antibody cross-reactivity. A key finding is that the epitope GRAFVTIG in the V3 loop of gp120 (strain IIIB) is homologous to the functionally essential U1 RNA-binding site of 70K. ELISA data reveal a mean reactivity of anti-RNP antibodies to V3 IIIB that is as high as that of HIV sera. V3 MN, containing the framework sequence G-AF-T, also cross-reacts with anti-RNP antibodies, as do hydrophilic epitopes in gp41 homologous to the COOH end of 70K. Further, there is strong cross-reactivity between HIV sera and 70K in Western blots. In contrast, antibodies from a related autoimmune disorder, Sjögren's syndrome (SS), are neither V3 nor gp41 selective. We conclude that the substantial cross-reactivities reported here are due to conserved, antigenically dominant B cell epitopes having homologous counterparts in 70K and gp120/41. Because antibody production in both MCTD and HIV-1 infection is T cell dependent, the results imply that common T cell clones are also activated in these two disease paradigms. Further exploration of the mechanisms that activate these clones, and that control their divergent fates in MCTD and AIDS, may provide new insights into immune dysregulation in HIV infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional activity of an HIV-1 neutralizing IgG human monoclonal antibody: ADCC and complement-mediated lysis.

The IgG1 kappa, human monoclonal antibody (HMAb), F105, was studied for functional activity in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). F105 reacts with a discontinuous epitope on the CD4 binding site of the HIV-1 envelope glycoprotein, gp120, expressed on the surfaces of infected cells and neutralizes diverse viral strains at antibody concentrations readily achievable in humans. Neither F105 nor serum (diluted 1:50) from HIV seropositive donors mediate CDC against an SF2-infected cell line with rabbit or human sera as a source of complement. F105 and HIV-1 sera mediate ADCC against the SF2 strain. Normal human serum reduced spontaneous lysis of SF2 by peripheral blood monocytes (PBM). Although mixing of F105 with normal human serum reduced the lysis observed (36 +/- 8 vs. 42 +/- 8%), this still was significantly greater than lysis in media (30 +/- 5%) or normal human serum (23 +/- 6%) (p less than .05). A murine antibody to CD16 significantly reduced spontaneous lysis observed with media (30 +/- 5 vs. 18 +/- 3%) while normal mouse serum had no effect (31 +/- 7%). ADCC mediated by F105 is completely abrogated by the anti-CD16 antibody (42 +/- 8 vs. 22 +/- 4%), while only a fraction of ADCC mediated by HIV sera is inhibited by anti-CD16 (60 +/- 9 vs. 46 +/- 6%), suggesting that several populations of effector cells function in ADCC mediated by the polyclonal sera. Thus, F105, as opposed to polyclonal sera, mediates ADCC through a CD16+ PBM population.

Biological and molecular variability of human immunodeficiency virus type 2 isolates from The Gambia

Seven new human immunodeficiency virus type 2 (HIV-2) isolates (CBL-20 to CBL-26) from The Gambia were characterized. Their cytopathogenicity and growth in vitro correlated with the severity of clinical disease. CBL-22 was highly sensitive to neutralization by HIV-2 sera and was cross-neutralized by some HIV-1 sera. These findings, the differing sizes of envelope glycoproteins of individual isolates, and the sequence analysis of amplified regions of the viral DNAs show that these HIV-2 isolates from one geographical region in West Africa exhibit biological and genome variability comparable to that observed for HIV-1.

Efficacies of US Food and Drug Administration-licensed HIV-1-screening enzyme immunoassays for detecting antibodies to HIV-2

To determine the efficacy of enzyme immunoassays (EIAs) for antibodies against HIV-1 in detecting HIV-2-infected blood, we tested 55 HIV-2-positive sera with seven Food and Drug Administration-licensed EIA kits. The percentage detection of HIV-2 sera giving positive reactions with these kits varied between the various manufacturers from 60 to 91%. Observations based on a small number of sera (n = 13), suggest that HIV-2-positive blood collected from apparently healthy people (blood donors, prenatal clinics) are detected with a greater frequency (means = 89%) than blood from AIDS patients or patients (n = 32) hospitalized with other infectious diseases (means = 72%). Based on these results and the low incidence of HIV-2 infection observed in the USA, it was concluded that screening with HIV-2-specific tests would not significantly increase the number of HIV-2-positive people detected by current screening programs. However, due to the poor sensitivity of certain HIV-1 assays for HIV-2 antibodies, HIV-2 sera without cross-reacting antibodies will escape detection. Surveillance for HIV-2 might then be improved by the availability of HIV-1 and HIV-2 combination assays.

Elevated plasma glutamate levels in colorectal carcinoma patients and in patients with acquired immunodeficiency syndrome (AIDS)

Amino acid concentrations were analyzed in the sera of HIV (LAV/HTLV-III) positive persons and in the plasma of colorectal carcinoma patients. Both groups of persons showed significantly elevated glutamate concentrations when compared with healthy control persons. Glutamate concentrations were found to be strongly elevated in all groups of HIV-positive persons including patients with acquired immunodeficiency syndrome (AIDS) or lymphadenopathy as well as HIV-positive persons without overt symptoms, indicating that increased plasma glutamate levels may be among the earliest consequences of the HIV infection. Moreover, the increased plasma glutamate concentrations in the colorectal carcinoma patients were correlated with a decreased immunological reactivity (mitogenic responses against concanavalin A). This suggests the possibility that the increased plasma glutamate concentrations may be causally responsible for the decreased immunological reactivity in colorectal carcinoma patients as well as in patients with AIDS.