Nadine Vincent

Identification of a conserved domain of the HIV-1 transmembrane protein gp41 which interacts with cholesteryl groups

A soluble form of the HIV-1 envelope glycoprotein gp160 devoid of the transmembrane anchor domain was found to bind to cholesteryl-hemisuccinate agarose. The external subunit gp120 failed to bind to the resin, suggesting that the site responsible for the binding to cholesterol was located in the transmembrane protein gp41. We constructed a series of maltose binding protein (MBP) fusion proteins representing overlapping fragments of the gp41 molecule and we studied their capacity to bind to cholesteryl beads. The domain responsible for binding to cholesterol was localised within the residues 668 to 684 immediately adjacent to the membrane spanning domain. We identified a short sequence (LWYIK, aa 678-683) comparable to the cholesterol interaction amino acid consensus pattern published by Li and Papadopoulos [Endocrinology 139 (1998) 4991]. We demonstrated that the sequence LWYIK synthesized fused to the MBP was able to bind to cholesteryl groups. A synthetic peptide containing the sequence LWYIK was found to inhibit the interaction between cholesteryl beads and MBP44, an MBP fusion HIV-1 envelope protein that contains the putative cholesterol binding domain. Human sera obtained from HIV-1 seropositive patients did not react in ELISA to the LWYIK sequence, suggesting that this region is not exposed to the immune system. The biological significance of the interaction between gp41 and cholesterol is discussed.

Detection of IgA inhibiting the interaction between gp120 and soluble CD4 receptor in serum and saliva of HIV-1-infected patients.

Objective: To evaluate the presence of IgA directed to the CD4-binding domain of gp120 and to a conserved region of gp41 (the Kennedy epitope) in serum and parotid saliva of HIV-1-seropositive patients. Methods: IgA were separated from IgG by anion-exchange chromatography and protein G treatment. The reactivity of IgA was tested against peptides and fusion proteins of the maltose-binding protein (MBP) and the CD4-binding site (MBP24) and MBP and the Kennedy epitope (MBP42). The capacity of serum and saliva IgA to interfere with the gp120–soluble CD4 (sCD4) interaction was examined. IgA were also purified by affinity chromatography using the MBP proteins adsorbed to a resin. Results: Peptides representing the CD4-binding domain and the Kennedy epitope were recognized by serum and saliva IgA of HIV-1-seropositive patients. Of the sera and saliva samples tested, 6/26 serum IgA and 5/25 saliva IgA inhibited the gp120–sCD4 interaction by approximately 50%. The gp120–sCD4 interaction was inhibited by MBP24 affinity-purified IgA but not by MBP42 affinity-purified IgA. Conclusion: Immunogens capable of eliciting IgA antibodies that inhibit gp120–CD4 binding might be efficiently used in vaccine to prevent mucosal transmission of HIV-1.

Antibodies purified from sera of HIV-1-infected patients by affinity on the heptad repeat region 1/heptad repeat region 2 complex of gp41 neutralize HIV-1 primary isolates.

Objective:The objective of this paper was to evaluate the presence and the neutralizing activity of antibodies directed against the complex formed between the two heptad repeat regions (HR1 and HR2) of HIV-1 gp41 in sera of HIV-1-infected patients. Research designs and methods:The HR1 region was represented by the peptide N36 and the maltose-binding protein (MBP)–HR1, the HR2 region by the peptide C34 and MBP44. Antibodies directed to the HR1/HR2 complex were purified from sera by affinity chromatography using MBP–HR1/C34 adsorbed onto a resin. Results:First, we demonstrated that human monoclonal antibodies, which are directed specifically to the HR1/HR2 complex recognized in enzyme-linked immunosorbent assay the MBP–HR1/C34 and MBP44/N36 mixtures but not the proteins or the peptides individually. We investigated the ability of 50 sera of HIV-1-infected patients to react with the MBP–HR1/C34 and MBP44/N36 complexes. We found that the majority of sera of HIV-1-infected patients recognized the HR1/HR2 complexes but not or to a lower extent the proteins or the peptides individually. Antibodies purified from sera by affinity chromatography using MBP–HR1/C34 adsorbed to a resin neutralized different primary HIV-1 isolates. Conclusion:The presence of antibodies directed to the HR1/HR2 complex in sera of HIV-infected patients highlights the immunogenic character of the complex, whereas the neutralizing activity of these antibodies suggests that immunogens representing HIV-1 HR1/HR2 complexes might be used in anti-HIV vaccine.

Simultaneous synthesis and degradation of glutamine in isolated rat liver cells. Effect of vasopressin

When hepatocytes suspensions obtained from whole livers of 48-h-fasted rats were incubated in Krebs-Henseleit buffer with a near-physiological concentration (1 mM) of L-[1-14C]glutamine as substrate, the apparent removal of glutamine was low, but the release of 14CO2 was much larger than the enzymatically measured removal of glutamine. This indicates that glutamine was metabolized at rates much higher than those accounted for by the apparent removal of glutamine. This also suggests that glutamine utilization was, at least in part, masked by concomitant synthesis of glutamine from endogenous substrates via glutamine synthetase. Evidence that such synthesis occurred was obtained by: (i) addition of methionine sulfoximine, an inhibitor of glutamine synthetase, which caused a large increase in the apparent removal of glutamine; and (ii) measurement of the specific radioactivity of L-[1-14C]glutamine which was shown to decrease during incubation. Addition of vasopressin (10(-7) M) led to a marked increase in glutamine removal by a dual mechanism: it accelerated flux through glutaminase, the enzyme which initiates the hepatic degradation of glutamine, and inhibited flux through glutamine synthetase.

Detection of AMP-activated protein kinase in human sera by immuno-isoelectric focusing.

AMPKalpha is a subunit of AMP-activated protein kinase (AMPK), a heterotrimeric enzyme that works as a fuel sensor activated in response to the depletion of cellular ATP. AMPKalpha is considered as a master switch in regulating glucose and lipid metabolism. Determining its presence in patient sera may help in diagnosing metabolic diseases. Using isoelectric focusing and Western blotting, we were able to detect AMPKalpha in human sera. Using specific antibodies, we showed that the AMPKalpha1 and alpha2 isoforms were apparently present in equal amounts in human sera. To characterize normal and abnormal AMPKalpha patterns, we used an antibody which recognized both isoforms (alpha1 and alpha2) to analyze sera of patients and healthy individuals. We also analyzed sera of HIV patients because several studies suggest that AMPK may play a role in the mechanism of lipodystrophy in HIV patients under antiretroviral therapy. We found that patients with type 2 diabetes or liver diseases presented abnormal AMPK IEF patterns. AMPK was poorly detectable in sera of patients with end-stage liver disease. Abnormal AMPK IEF patterns were more frequent in treated HIV-patients compared to those who are untreated suggesting a possible association between AMPK and the side-effects of antivirals. Our findings highlight the potential of serum AMPK as a new diagnostic biomarker and may help to study the regulation of AMPK activity in tissues.

Opposite immune reactivity of serum IgG and secretory IgA to conformational recombinant proteins mimicking V1/V2 domains of three different HIV type 1 subtypes depending on glycosylation.

The V1/V2 domain of the HIV-1 gp120 envelope protein has been shown to contribute to viral cell tropism during infection and also to viral recognition by neutralizing monoclonal antibodies. However, this domain has been poorly investigated. Carbohydrates have been demonstrated to dramatically influence immune reactivity of antisera to viral glycoprotein antigens. In this study, DNA sequences coding for V1/V2 domains from HIV-1 primary isolates of three subtypes (A, B, and C) were subcloned into a secretion vector and used to transfect CHO cells that are able to achieve the glycosylation of proteins. The structure of purified recombinant V1/V2 proteins was tested using two anti-V1/V2 monoclonal antibodies directed against either a linear or a conformational and glycosylation-dependent epitope (8.22.2 and 697-D). Serum or saliva of 14/82 seropositive patients with anti-V1/V2 reactivity demonstrated good recognition of the recombinant proteins. Deglycosylation of the recombinant proteins was found to increase the reactivity of the serum IgG to the clade A and C but not to clade B V1/V2 domain demonstrating that the recognition of glycosylation sites by serum IgG is clade dependent. When considering SIgA from parotid saliva, deglycosylation of all recombinant proteins tested decreased the reactivity, suggesting that glycosylation plays an important role in the recognition of V1/V2 domain target epitopes by this class of antibodies. In conclusion, these results suggest the influence of carbohydrate moieties on the specificity of the antibodies to the V1/V2 domain produced during HIV infection and the potential importance of viral glycans in vaccine responses after mucosal administration.

Ability of antibodies specific to the HIV-1 envelope glycoprotein to block the fusion inhibitor T20 in a cell–cell fusion assay

The anti-HIV peptide T20 is able to inhibit the syncytia formation between CHO-WT and HeLa CD4(+)cells. We found that several sera of HIV-infected patients have the capacity to block the inhibition of fusion by T20. Suggesting that these sera may contain antibody which can block T20 access and prevent membrane fusion, we studied the ability of a panel of antibodies directed to different regions of HIV-1 envelope glycoprotein to block the inhibition of fusion by T20. We found that the C1 and V3 loop regions of gp120 and the heptad repeat 1, the immunodominant C-C region and the Kennedy epitope of gp41 located in the intracytoplasmic tail were the target for antibodies capable to block the inhibition of syncytia formation by T20. We suggest that these antibodies have the capacity to counteract the anti-fusion effect of T20 by preventing its binding to the interaction sites. Further studies are needed to determine if some of them recognize new T20 interaction sites.

Soluble chemokine receptor CXCR4 is present in human sera

A soluble form of the chemokine receptor CXCR4 was detected in human sera by isoelectric focusing and Western blotting. Sera of patients and normal subjects were analyzed using a panel of specific antibodies. Compared with controls, high levels of serum CXCR4 were found in patients with inflammatory bowel diseases. Serum CXCR4 levels in the majority of HIV patients were similar to those in healthy controls. A sensitive polyclonal antibody was developed in rabbit immunized with a maltose binding protein (MBP) construct expressing the full-length CXCR4. Using anti-MBPCXCR4 antibody, the level of CXCR4 in sera of a majority of patients with fibrosis was very low. The potential of serum CXCR4 as a new diagnostic biomarker warrants further investigation.

Hyperglycosylated ferritin in sera of HIV-1-infected patients treated with highly active antiretroviral therapy

A study was conducted to determine the relationship between ferritin and glycosylated isoforms of ferritin and insulin resistance in 69 HIV-infected men receiving HAART. Ferritin levels were significantly correlated with aspartate aminotransferase, alanine aminotransferase, bilirubin and with insulin resistance. The ferritin isoelectric focusing patterns of five insulin-resistant HIV-infected patients under HAART showed large amounts of hyperglycosylated isoforms, which was not found in 56 control subjects and 46 untreated HIV-1-infected patients.

First Membrane Proximal External Region–Specific Anti-HIV1 Broadly Neutralizing Monoclonal IgA1 Presenting Short CDRH3 and Low Somatic Mutations

Mucosal HIV-1–specific IgA have been described as being able to neutralize HIV-1 and to block viral transcytosis. In serum and saliva, the anti-HIV IgA response is predominantly raised against the envelope of HIV-1. In this work, we describe the in vivo generation of gp41-specific IgA1 in humanized α1KI mice to produce chimeric IgA1. Mice were immunized with a conformational immunogenic gp41-transfected cell line. Among 2300 clones screened by immunofluorescence microscopy, six different gp41-specific IgA with strong recognition of gp41 were identified. Two of them have strong neutralizing activity against primary HIV-1 tier 1, 2, and 3 strains and present a low rate of somatic mutations and autoreactivity, unlike what was described for classical gp41-specific IgG. Epitopes were identified and located in the hepted repeat 2/membrane proximal external region. These Abs could be of interest in prophylactic treatment to block HIV-1 penetration in mucosa or in chronically infected patients in combination with antiretroviral therapy to reduce viral load and reservoir.