Human Immunodeficiency Virus Type 1 Elite Neutralizers: Individuals with Broad and Potent Neutralizing Activity Identified by Using a High-Throughput Neutralization Assay together with an Analytical Selection Algorithm

The development of a rapid and efficient system to identify human immunodeficiency virus type 1 (HIV-1)-infected individuals with broad and potent HIV-1-specific neutralizing antibody responses is an important step toward the discovery of critical neutralization targets for rational AIDS vaccine design. In this study, samples from HIV-1-infected volunteers from diverse epidemiological regions were screened for neutralization responses using pseudovirus panels composed of clades A, B, C, and D and circulating recombinant forms (CRFs). Initially, 463 serum and plasma samples from Australia, Rwanda, Uganda, the United Kingdom, and Zambia were screened to explore neutralization patterns and selection ranking algorithms. Samples were identified that neutralized representative isolates from at least four clade/CRF groups with titers above prespecified thresholds and ranked based on a weighted average of their log-transformed neutralization titers. Linear regression methods selected a five-pseudovirus subset, representing clades A, B, and C and one CRF01_AE, that could identify top-ranking samples with 50% inhibitory concentration (IC₅₀) neutralization titers of ≥100 to multiple isolates within at least four clade groups. This reduced panel was then used to screen 1,234 new samples from the Ivory Coast, Kenya, South Africa, Thailand, and the United States, and 1% were identified as elite neutralizers. Elite activity is defined as the ability to neutralize, on average, more than one pseudovirus at an IC₅₀ titer of 300 within a clade group and across at least four clade groups. These elite neutralizers provide promising starting material for the isolation of broadly neutralizing monoclonal antibodies to assist in HIV-1 vaccine design.Since the identification of human immunodeficiency virus type 1 (HIV-1) as the cause of AIDS, one of the greatest challenges has been the development of a vaccine that will prevent infection and/or ameliorate disease progression (38, 43). Although over 100 phase I, II, and III vaccine clinical trials of different candidates have been conducted all over the world, only a few candidates have advanced to efficacy testing and none has yet to show any benefit in prevention or control of HIV-1 (HIV Vaccine Database; www.iavi.org). In other viral diseases (such as polio, influenza, and measles), neutralizing antibodies are generated as part of either the natural immune response to infection or the response to immunization, and their role in protective immunity is well established (10, 12, 15, 22, 37, 42, 45, 47, 49, 52). For HIV-1, studies in animal models indicate that both broadly neutralizing antibodies and cell-mediated responses may be required to provide vaccine protection (7, 14, 16, 20, 29, 31, 33, 34, 39, 53). Unlike many other viruses, HIV-1 is highly variable, with multiple subtypes and recombinant forms circulating in different regions of the world. This high level of HIV-1 genetic variability, particularly in the envelope glycoproteins (gp120 and gp41), has been one of the greatest obstacles in development of a safe and effective HIV-1 vaccine and in particular in the elicitation of broadly neutralizing antibodies. In addition, HIV-1 has other mechanisms of immune escape preventing elicitation of broadly neutralizing antibodies, including the heavy glycosylation of the envelope glycoproteins, instability of such glycoproteins, and conformational masking of receptor-binding sites (6, 25, 32).Despite the enormous diversity of HIV-1, a relatively small number of broadly neutralizing monoclonal antibodies (bnMAbs) have been isolated, providing evidence that broad neutralization by single antibody specificities can be achieved (3-5, 8, 9, 17, 21, 23, 24, 29, 35, 36, 40, 41, 44, 50, 51, 55). Structures for such bnMAbs have been determined in complex with HIV-1 Env (26, 54) and provide starting points for the design of immunogens capable of eliciting broadly neutralizing antibodies. However, since there are only a few such bnMAbs, we established a global program as part of International AIDS Vaccine Initiative''s (IAVI''s) Neutralizing Antibody Consortium (6), aimed at screening HIV-1⁺ subjects with the goal of identifying individuals with broad and potent neutralizing activities as a potential source of novel bnMAbs, with an emphasis placed on individuals infected with non-clade B viruses. This paper describes the screening algorithm implemented to successfully identify HIV-1⁺ subjects with broadly neutralizing antibodies, including a subset of individuals termed “elite neutralizers.” These volunteers will be studied further to characterize the specificities of serum antibodies and will provide source materials for isolation of bnMAbs.     

Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies from an HIV-1 Vaccine Efficacy Trial Target Multiple Epitopes and Preferentially Use the VH1 Gene Family

The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine trial showed an estimated efficacy of 31%. RV144 secondary immune correlate analysis demonstrated that the combination of low plasma anti-HIV-1 Env IgA antibodies and high levels of antibody-dependent cellular cytotoxicity (ADCC) inversely correlate with infection risk. One hypothesis is that the observed protection in RV144 is partially due to ADCC-mediating antibodies. We found that the majority (73 to 90%) of a representative group of vaccinees displayed plasma ADCC activity, usually (96.2%) blocked by competition with the C1 region-specific A32 Fab fragment. Using memory B-cell cultures and antigen-specific B-cell sorting, we isolated 23 ADCC-mediating nonclonally related antibodies from 6 vaccine recipients. These antibodies targeted A32-blockable conformational epitopes (n = 19), a non-A32-blockable conformational epitope (n = 1), and the gp120 Env variable loops (n = 3). Fourteen antibodies mediated cross-clade target cell killing. ADCC-mediating antibodies displayed modest levels of V-heavy (VH) chain somatic mutation (0.5 to 1.5%) and also displayed a disproportionate usage of VH1 family genes (74%), a phenomenon recently described for CD4-binding site broadly neutralizing antibodies (bNAbs). Maximal ADCC activity of VH1 antibodies correlated with mutation frequency. The polyclonality and low mutation frequency of these VH1 antibodies reveal fundamental differences in the regulation and maturation of these ADCC-mediating responses compared to VH1 bNAbs.     

Comparative study of controlled pore glass, silica gel and poraver for the immobilization of urease to determine urea in a flow injection conductimetric biosensor system

This study compared the responses of three enzyme reactors containing urease immobilized on three types of solid support, controlled pore glass (CPG), silica gel and Poraver. The evaluation of each enzyme reactor column was done in a flow injection conductimetric system. When urea in the sample solution passed though the enzyme reactor, urease catalysed the hydrolysis of urea into charged products. A lab-built conductivity meter was used to measure the increase in conductivity of the solution. The responses of the enzyme reactor column with urease immobilized on CPG and silica gel were similar and were much higher than that of Poraver. Both CPG and silica gel reactor columns gave the same limit of detection, 0.5 mM, and the response was still linear up to 150mM. The analysis time was 4-5 min per sample. The enzyme reactor column with urease immobilized on CPG gave a slightly better sensitivity, 4% higher than the reactor with silica gel. The life time of the immobilized urease on CPG and silica gel were more than 310h operation time (used intermittently over 7 months). Good agreement was obtained when urea concentrations of human serum samples determined by the flow injection conductimetric biosensor system was compared to the conventional methods (Fearon and Berthelot reactions). These were statistically shown using the regression line and Wilcoxon signed rank tests. The results showed that the reactor with urease immobilized on silica gel had the same efficiency as the reactor with urease immobilized on CPG.     

Juvenile hormone acid methyltransferase activity in imaginal discs of Manduca sexta prepupae

The occurrence of a peak of juvenile hormone (JH) during the prepupal period has been noted in several lepidopterans. In Manduca sexta and Hyalophora cecropia this peak is known to prevent the precocious onset of adult differentiation in imaginal tissues. However, it has previously been observed in our laboratory that corpora allata (CA) of this age are incapable of making JH owing to a lack of the terminal synthetic enzyme, juvenile hormone acid methyltransferase (JHAMT). Since the CA are required for normal pupation, it is likely that JH acid is the product released by the prepupal CA. Therefore, we analyzed whether JH acid treatment would prevent precocious adultoid differentiation in allatectomized M. sexta larvae. JH acid injections were found to be as effective as JH in normalizing pupation, and acted in a time- and dose-dependent manner. This finding led to a question of whether injected or endogenous JH acid could be methylated to JH. Homogenates of several tissues from prepupae were assayed for the presence of JHAMT. Of the tissues assayed, only imaginal discs possessed significant levels of the enzyme. These results support our previously proposed mechanism for production of the prepupal JH peak in M. sexta.     

Isolation of a Pandemic O3:K6 Clone of a Vibrio parahaemolyticus Strain from Environmental and Clinical Sources in Thailand

Application of an immunomagnetic enrichment method selective for Vibrio parahaemolyticus serovar K6 allowed isolation of a strain belonging to the pandemic O3:K6 clone of V. parahaemolyticus from fresh shellfish not implicated in a clinical case in southern Thailand. Arbitrarily primed PCR profiles of this strain, clinical O3:K6 strains isolated from sporadic diarrhea cases in the same area, and a standard pandemic O3:K6 strain were indistinguishable.     

Selecting participants fairly for controlled human infection studies

Controlled human infection (CHI) studies involve the deliberate exposure of healthy research participants to infectious agents to study early disease processes and evaluate interventions under controlled conditions with high efficiency. Although CHI studies expose participants to the risk of infection, they are designed to offer investigators unique advantages for studying the pathogenesis of infectious diseases and testing potential vaccines or treatments in humans. One of the central challenges facing investigators involves the fair selection of research subjects to participate in CHI studies. While there is widespread agreement that investigators have a duty to select research participants fairly, this principle also yields conflicting ethical imperatives, for example requiring investigators to both exclude potential participants with co-morbidities since they face increased risks, but also to include them in order to ensure generalizability. In this paper we defend an account of fair subject selection that is tailored to the context of CHI studies. We identify the considerations of fairness that bear directly on selecting participants for CHI studies and provide investigators and members of IRBs and RECs with a principled way to navigate the conflicting imperatives to which these considerations give rise.     

The Thai phase III trial (RV144) vaccine regimen induces T cell responses that preferentially target epitopes within the V2 region of HIV-1 envelope

The Thai HIV phase III prime/boost vaccine trial (RV144) using ALVAC-HIV (vCP1521) and AIDSVAX B/E was, to our knowledge, the first to demonstrate acquisition efficacy. Vaccine-induced, cell-mediated immune responses were assessed. T cell epitope mapping studies using IFN-γ ELISPOT was performed on PBMCs from HIV-1-uninfected vaccine (n = 61) and placebo (n = 10) recipients using HIV-1 Env peptides. Positive responses were measured in 25 (41%) vaccinees and were predominantly CD4(+) T cell-mediated. Responses were targeted within the HIV Env region, with 15 of 25 (60%) of vaccinees recognizing peptides derived from the V2 region of HIV-1 Env, which includes the α(4)β(7) integrin binding site. Intracellular cytokine staining confirmed that Env responses predominated (19 of 30; 63% of vaccine recipients) and were mediated by polyfunctional effector memory CD4(+) T cells, with the majority of responders producing both IL-2 and IFN-γ (12 of 19; 63%). HIV Env Ab titers were higher in subjects with IL-2 compared with those without IL-2-secreting HIV Env-specific effector memory T cells. Proliferation assays revealed that HIV Ag-specific T cells were CD4(+), with the majority (80%) expressing CD107a. HIV-specific T cell lines obtained from vaccine recipients confirmed V2 specificity, polyfunctionality, and functional cytolytic capacity. Although the RV144 T cell responses were modest in frequency compared with humoral immune responses, the CD4(+) T cell response was directed to HIV-1 Env and more particularly the V2 region.     

Comparison of surface plasmon resonance and capacitive immunosensors for cancer antigen 125 detection in human serum samples

This paper presents a comparison between surface plasmon resonance (SPR) and capacitive immunosensors for a flow injection label-free detection of cancer antigen 125 (CA 125) in human serum. Anti-CA 125 was immobilized on gold surface through a self-assembled monolayer. Parameters affecting the responses of each system were optimized. Under optimal conditions, SPR provided a detection limit of 0.1 U ml⁻¹ while 0.05 U ml⁻¹ was obtained for the capacitive system. Linearity for SPR was between 0.1 and 40 U ml⁻¹ and 0.05–40 U ml⁻¹ for capacitive system. These immunosensors were applied to analyze CA 125 concentrations in human serum samples and compared with conventional enzyme linked fluorescent assay (ELFA). Both systems showed good agreement with ELFA (P < 0.05). Moreover, these immunosensors were very stable and provided good reproducible responses after regeneration, up to 32 times for SPR and 48 times for capacitive system with relative standard deviation lower than 4%. The SPR immunosensor provided advantages in term of fast response and real-time monitoring while capacitive immunosensor offered a sensitive and cost-effective method for CA 125 detection.     

Change in corpus allatum function during metamorphosis of the tobacco hornworm Manduca sexta: Regulation at the terminal step in juvenile hormone biosynthesis

Changes in activity of the corpora allata (CA) during larval-pupal-adult development of the tobacco hornworm Manduca sexta were studied by transplantation assays, measurements of in vitro juvenile hormone (JH) and JH acid synthesis, and determination of JH acid methyltransferase (JHAMT) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activities. The data from these assays demonstrate that the CA cease to secrete JH by day 4 of the last larval instar (wandering stage). With regard to JH synthesis, they remain inactive throughout the prepupal, pupal, and most of the pharate adult periods. CA of females, but not of males, resume JH synthesis shortly before eclosion. The biochemical basis of the inactivation process is the loss of JHAMT activity. However, prepupal CA produce JH acids, as shown by enzyme and in vitro assays. Pupal and pharate adult CA do not synthesize JH acids although levels of HMG-CoA reductase activity seem to remain relatively high. Radiolabeled JH was recovered from hemolymph of allatectomized prepupae that had been injected with radiolabeled JH acid. These results provide further evidence that certain peripheral tissues (eg, imaginal discs) convert JH acid secreted by the prepupal CA to JH and, thus, that JH acid is a prohormone in the prepupal period. The CA change from hormone secretion to prohormone secretion during larval-prepupal transformation, a unique functional alteration in an endocrine gland.