Jens Ole Nielsen

Correlation between carbohydrate structures on the envelope glycoprotein gp120 of HIV-1 and HIV-2 and syncytium inhibition with lectins.

The binding of 13 different lectins to gp120 partially purified from two HIV-1 isolates and one HIV-2 isolate was studied by in situ staining on electrophoretically separated and electroblotted HIV antigens. The lectins concanavalin A, wheat germ agglutinin, Lens culinaris agglutinin, Vicia faba agglutinin, Pisum sativum agglutinin and phytohaem(erythro)agglutinin bound to gp120 of all three isolates. The carbohydrate of gp120 recognized by lectins was thus arranged in at least four types of glycans: a high mannose type glycan, a bisected hybrid or complex type glycan, a biantennary fucosylated complex type glycan and a triantennary bisected complex type glycan. Only lectins which bound at least one of the four types of glycans were capable of inhibiting fusion of HIV-infected cells with CD4 cells by a carbohydrate-specific interaction with the HIV-infected cells. Thus, several different glycan structures may be implicated in CD4-gp120 binding.

Association between Larger Thymic Size and Higher Thymic Output in Human Immunodeficiency Virus-Infected Patients Receiving Highly Active Antiretroviral Therapy

To examine the impact of thymic size on immune recovery in patients with human immunodeficiency virus (HIV) infection, the thymus was visualized, using computed tomographic scans, in 25 HIV-infected patients who had received highly active antiretroviral therapy (HAART) for 6-18 months and had levels of viremia <500 copies/mL. For comparison, 10 control subjects were included in the study. Total and naive CD4+ cell counts were determined by flow cytometry. To determine thymic output, the number of CD4+ cells containing T cell receptor excision circles (TRECs) was measured. Qualitative immune recovery was evaluated by determination of CD4+ T cell receptor repertoire in 19 of the HIV-infected patients. Larger thymic size was associated with higher CD4+ cell counts (r=0.498; P=.011) and higher CD4+ TREC frequency (r=0.652; P<.001). Furthermore, patients with abundant thymic tissue seemed to have broader immunologic repertoires, compared with patients with minimal thymic tissue (P=.054). These findings suggest that thymopoiesis is ongoing in the adult thymus and contributes to immune reconstitution in HIV-infected patients receiving HAART.

Hepatitis type A, B, and non-A non-B in fulminant hepatitis.

Serological investigations for hepatitis B surface and e antigen, antibody to hepatitis B surface, core and e antigen and antibody to hepatitis A virus were carried out in 22 patients with fulminant hepatitis admitted to Medical Department A, Rigshospitalet, Copenhagen, in 1970-77. Nine patients had hepatitis type B and four type A. One patient had evidence of both type A and B infection, whereas the remaining eight patients showed no evidence of type A or B infection. Two of these had been treated with disulfiram and a drug aetiology could not be excluded, but in six patients no known cause of fulminant hepatitis could be determined and these patients were classified as having hepatitis type non-A non-B. The survival rate was not statistically different for patients having type A, B, or non-A non-B hepatitis.

Cerebral atrophy in AIDS: a stereological study

SummaryStereological estimates of mean volumes, surface areas, and cortical thicknesses were obtained on formalin-fixed brains from 19 men with AIDS and 19 controls. Volumes of neocortex, white matter, central brain nuclei, ventricles and archicortex were estimated using point counting and Cavalieris unbiased principle for volume estimation. In AIDS, the mean volume of neocortex was reduced by 11%, and that of the central brain nuclei by 18%. Mean ventricular volume was increased by 55%. Mean neocortical thickness was reduced by 12%. The mean volume of white matter was reduced by 13%. The findings in 6 clinically demented AIDS patients were not statistically different from the rest of the group.

An O-linked carbohydrate neutralization epitope of HIV-1 gp 120 is expressed by HIV-1 env gene recombinant vaccinia virus

SummaryPrevious studies have disagreed about the presence of O-linked carbohydrate epitopes on gp 120 of HIV, although antibodies against short-chain O-linked glycans neutralize HIV infection and block syncytium formation in vitro. To settle this question, we analysed the O-linked glycans of gp 120 by chemical methods using purified HIV-1 gp 120 from cells infected with recombinant vaccinia virus solely expressing gp 160 or gp 120. Alkaline borohydride degradation of recombinant gp 120 released monosaccharides and also slightly larger structures (di/trisaccharides) by a β-elimination, confirming the presence of simple O-linked oligosaccharides. The functional activity as neutralisation epitopes of the O-linked oligosaccharides expressed on recombinant gp 120 was preserved, since fusion between uninfected CD 4+ cells and cells infected with recombinant vaccinia was blocked by monoclonal antibodies to the O-linked oligosaccharides of gp 120. Although the mechanism for HIV induction of O-linked oligosaccharide neoantigens is unknown, these results indicate that the O-linked neutralization epitopes are inherent to the glycoprotein itself, and that the unusual appearance of simple O-linked oligosaccharides on gp 120 is independent of any interaction between the host cell and retroviral genes other thanenv.

Identification of an N-linked glycan in the V1-loop of HIV-1 gp120 influencing neutralization by anti-V3 antibodies and soluble CD4.

SummaryGlycosylation is necessary for HIV-1 gp120 to attain a functional conformation, and individual N-linked glycans of gp120 are important, but not essential, for replication of HIV-1 in cell culture. We have constructed a mutant HIV-1 infectious clone lacking a signal for N-linked glycosylation in the V1-loop of HIV-1 gp120. Lack of an N-linked glycan was verified by a mobility enhancement of mutant gp120 in SDS-gel electrophoresis. The mutated virus showed no differences in either gp120 content per infectious unit or infectivity, indicating that the N-linked glycan was neither essential nor affecting viral infectivity in cell culture. We found that the mutated virus lacking an N-linked glycan in the V1-loop of gp120 was more resistant to neutralization by monoclonal antibodies to the V3-loop and neutralization by soluble recombinant CD4 (sCD4). Both viruses were equally well neutralized by ConA and a conformation dependent human antibody IAM-2G12. This suggests that the N-linked glycan in the V1-loop modulates the three-dimensional conformation of gp120, without changing the overall functional integrity of the molecule.

Expression of the activation antigen CD69 predicts functionality of in vitro expanded peripheral blood mononuclear cells (PBMC) from healthy donors and HIV-infected patients

Gene therapy for AIDS necessitates harvest and expansion of PBMC from HIV‐infected patients. We expanded PBMC from healthy blood donors and HIV‐infected patients for up to 14 days using four expansion protocols: 3 days of phytohaemagglutinin (PHA) stimulation, continuous PHA stimulation, 3 days of stimulation with anti‐CD3 and anti‐CD28, and continuous stimulation with anti‐CD3 and anti‐CD28. Functionality of PBMC was evaluated prior to and after expansion using standard proliferation assay. Phenotype and lymphocyte subset activation defined by expression of CD69 and CD25 were determined using flow cytometry. PBMC from healthy donors and HIV‐infected patients were readily expanded. The best expansion was obtained using stimulation for 3 days. After expansion, functionality of PBMC measured as proliferative response was partly conserved. PBMC expanded with stimulation for 3 days exhibited more preserved functionality than PBMC stimulated continuously (Pu2003<u20030.03). The mean proliferative response in each of the four different expansion protocols correlated with the mean values of CD69 expression. The proliferative responses from patients and healthy donors expanded with PHA stimulation for 3 days correlated with CD69 expression on CD4 cells (ru2003=u20030.68, Pu2003<u20030.01) and on CD8 cells (ru2003=u20030.59, Pu2003<u20030.03). Furthermore, expression of CD69 reliably predicted which patients and donors had highly conserved functionality after in vitro expansion. Finally, PBMC expanded with PHA stimulation for 3 days were examined for apoptosis. Only a minor fraction was primed for apoptosis, and this fraction could be significantly reduced by addition of IL‐2 to the culture medium (Pu2003<u20030.05). In conclusion, the feasibility of expanding PBMC from HIV patients was demonstrated. Expanded PBMC had conserved functionality. Finally, after in vitro expansion, expression of the activation antigen CD69 reliably predicted functionality of PBMC.

Sensitivity of HIV-1 to neutralization by antibodies against O-linked carbohydrate epitopes despite deletion of O-glycosylation signals in the V3 loop

SummaryIt has been suggested that threonine or serine residues in the V3 loop of HIV-1 gp120 are glycosylated with the short-chain O-linked oligosaccharides Tn or sialosyl-Tn that function as epitopes for broadly neutralizing carbohydrate specific antibodies. In this study we examined whether mutation of such threonine or serine residues could decrease the sensitivity to infectivity inhibition by Tn or sialosyl-Tn specific antibodies. All potentially O-glycosylated threonine and serine residues in the V3 loop of cloned HIV-1BRU were mutagenized to alanine thus abrogating any O-glycosylation at these sites. Additionally, one of these T-A mutants (T308A) also abrogated the signal for N-glycosylation at N306 inside the V3-loop. The mutant clones were compared with the wild type virus as to sensitivity to neutralization with monoclonal and polyclonal antibodies specific for the tip of the V3 loop of BRU or for the O-linked oligosaccharides Tn or sialosyl-Tn. Deletion of the N-linked oligosaccharide at N306 increased the neutralization sensitivity to antibodies specific for the tip of the loop, which indicates that N-linked glycosylation modulates the accessibility to this immunodominant epitope. However, none of the mutants with deletions of O-glycosylation signals in the V3 loop displayed any decrease in sensitivity to anti-Tn or anti-sialosyl-Tn antibody. This indicates that these broadly specific neutralization epitopes are located outside the V3 loop of gp120.

Trends in survival of Danish AIDS patients from 1981 to 1989

Length of survival was analysed in relation to year of diagnosis, AIDS-indicative disease, age, risk behaviour, zidovudine therapy, and CD4 cell count and serum immunoglobulin (Ig) levels at the time of diagnosis in a group of 231 consecutive adult Danish AIDS patients reported before 1 January 1988. The cumulative survival rate was 53% (95% confidence interval 47-59%) at 1 year, 29% (22-36%) at 2 years and 18% (10-26%) at 3 years. Length of survival increased significantly (P less than 0.001) over time for patients who were initially diagnosed with Pneumocystis carinii pneumonia (PCP), 17% (3-31%) at 2 years prior to 1986, 32% (16-49%) in 1986 and 52% (34-69%) in 1987, whereas survival remained stable for patients with other AIDS-indicative diseases. Survival was similar for patients who were diagnosed with Kaposis sarcoma alone and PCP alone. Independent predictors of a shortened survival were a CD4 cell count less than 200 x 10(6)/l, a serum IgA level greater than 4 g/l, and an initial diagnosis with opportunistic infections other than PCP. In addition, the multivariate analysis suggested an improved survival in recent years for patients diagnosed with PCP, independent of other factors examined. We conclude that length of survival in AIDS patients is highly variable. Determinants of progression include CD4 cell count, serum IgA level, and presenting disease. Survival has increased markedly for patients with PCP and median survival now exceeds 24 months.

Six billion neurons lost in AIDS

Human immunodeficiency virus type 1 (HIV1) is neurotropic. One of the morphological changes that is seen in patients with acquired immunodeficiency syndrome (AIDS) is cerebral atrophy affecting various structures including the neocortex. The cause of atrophy is not known. The total number of neocortical neurons was estimated in formalin fixed brains of 12 males with AIDS and 12 male controls matched for age and height. The mean number of neocortical neurons was 16.0times109 (coefficient of variation=0.11) in the AIDS patients compared with 21.9times109 (coefficient of variation=0.22) in the controls, a difference of approximately six billion (p<0.005, 2‐tailed). The global neuronal loss was 37%, and affected all four neocortical lobes. Ten patients did not have a history of central nervous system symptoms; two patients had a history of dementia. The number of neurons in the AIDS cases was not associated with dementia. AIDS is the first disease in which a global loss of neocortical neurons has been demonstrated using unbiased stereological methods. The loss of more than one third of the neurons may partly explain the cortical atrophy. Focal neuron loss has been reported by several authors, but none have been based on unbiased methods. In this group of AIDS patients the severe loss of neurons did not correspond to neurological deficits.