Antigenic diversity thresholds and the development of AIDS.

Longitudinal studies of patients infected with HIV-1 reveal a long and variable incubation period between infection and the development of AIDS. Data from a small number of infected patients show temporal changes in the number of genetically distinct strains of the virus throughout the incubation period, with a slow but steady rise in diversity during the progression to disease. A mathematical model of the dynamic interaction between viral diversity and the human immune system suggests the existence of an antigen diversity threshold, below which the immune system is able to regulate viral population growth but above which the virus population induces the collapse of the CD4+ lymphocyte population. The model suggests that antigenic diversity is the cause, not a consequence, of immunodeficiency disease. The model is compared with available data, and is used to assess how the timing of the application of chemotherapy or immunotherapy influences the rate of progress to disease.

Phosphate-methylated DNA aimed at HIV-1 RNA loops and integrated DNA inhibits viral infectivity.

Phosphate-methylated DNA hybridizes strongly and specifically to natural DNA and RNA. Hybridization to single-stranded and double-stranded DNA leads to site-selective blocking of replication and transcription. Phosphate-methylated DNA was used to interrupt the life cycle of the human immunodeficiency virus type-1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS). Both antisense and sense phosphate-methylated DNA 20-nucleotide oligomers, targeted at the transactivator responsive region and the primer binding site, caused complete inhibition of viral infectivity at a low concentration. Hybridization of phosphate-methylated DNA with folded and unfolded RNA was studied by ultraviolet and proton nuclear magnetic resonance spectroscopy. The combined results of hybridization studies and biological experiments suggest that the design of effective antisense phosphate-methylated DNA should focus on hairpin loop structures in the viral RNA. For sense systems, the 5' end of the integrated viral genome is considered to be the important target site.

T cell telomere length in HIV-1 infection: no evidence for increased CD4+ T cell turnover.

Progression to acquired immunodeficiency syndrome (AIDS) has been related to exhaustion of the regenerative capacity of the immune system resulting from high T cell turnover. Analysis of telomeric terminal restriction fragment (TRF) length, a marker for cellular replicative history, showed that CD8(+) T cell TRF length decreased but CD4(+) T cell TRF length was stable during the course of human immunodeficiency virus type-1 (HIV-1) infection, which was not explained by differential telomerase activity. This observation provides evidence that turnover in the course of HIV-1 infection can be increased considerably in CD8(+) T cells, but not in CD4(+) T cells. These results are compatible with CD4(+) T cell decline in HIV-1 infection caused by interference with cell renewal.

Kinetics of response in lymphoid tissues to antiretroviral therapy of HIV-1 infection.

In lymphoid tissue, where human immunodeficiency virus-type 1 (HIV-1) is produced and stored, three-drug treatment with viral protease and reverse transcriptase inhibitors markedly reduced viral burden. This was shown by in situ hybridization and computerized quantitative analysis of serial tonsil biopsies from previously untreated adults. The frequency of productive mononuclear cells (MNCs) initially diminished with a half-life of about 1 day. Surprisingly, the amount of HIV-1 RNA in virus trapped on follicular dendritic cells (FDCs) decreased almost as quickly. After 24 weeks, MNCs with very few copies of HIV-1 RNA per cell were still detectable, as was proviral DNA; however, the amount of FDC-associated virus decreased by >/=3.4 log units. Thus, 6 months of potent therapy controlled active replication and cleared >99.9 percent of virus from the secondary lymphoid tissue reservoir.

Serum-free transient protein production system based on adenoviral vector and PER.C6 technology: high yield and preserved bioactivity.

Stable E1 transformed cells, like PER.C6, are able to grow at scale and to high cell densities. E1-deleted adenoviruses replicate to high titer in PER.C6 cells whereas subsequent deletion of E2A from the vector results in absence of replication in PER.C6 cells and drastically lowers the expression of adenovirus proteins in such cells. We therefore considered the use of an DeltaE1/DeltaE2 type 5 vector (Ad5) to deliver genes to PER.C6 cells growing in suspension with the aim to achieve high protein yield. To evaluate the utility of this system we constructed DeltaE1/DeltaE2 vector carrying different classes of protein, that is, the gene coding for spike protein derived from the Coronavirus causing severe acute respiratory syndrome (SARS-CoV), a gene coding for the SARS-CoV receptor or the genes coding for an antibody shown to bind and neutralize SARS-CoV (SARS-AB). The DeltaE1/DeltaE2A-vector backbones were rescued on a PER.C6 cell line engineered to constitutively over express the Ad5 E2A protein. Exposure of PER.C6 cells to low amounts (30 vp/cell) of DeltaE1/DeltaE2 vectors resulted in highly efficient (>80%) transduction of PER.C6 cells growing in suspension. The efficient cell transduction resulted in high protein yield (up to 60 picogram/cell/day) in a 4 day batch production protocol. FACS and ELISA assays demonstrated the biological activity of the transiently produced proteins. We therefore conclude that DeltaE1/DeltaE2 vectors in combination with the PER.C6 technology may provide a viable answer to the increasing demand for high quality, high yield recombinant protein.

Immunological abnormalities in human immunodeficiency virus (HIV)-infected asymptomatic homosexual men. HIV affects the immune system before CD4+ T helper cell depletion occurs.

To investigate the effect of persistent HIV infection on the immune system, we studied leukocyte functions in 14 asymptomatic homosexual men (CDC group II/III) who were at least two years seropositive, but who still had normal numbers of circulating CD4+ T cells. Compared with age-matched heterosexual men and HIV-negative homosexual men, the CD4+ and CD8+ T cells from seropositive men showed decreased proliferation to anti-CD3 monoclonal antibody and decreased CD4+ T-helper activity on PWM-driven differentiation of normal donor B cells. Monocytes of HIV-infected homosexual men showed decreased accessory function on normal T cell proliferation induced by CD3 monoclonal antibody. The most striking defect in leukocyte functional activities was observed in the B cells of HIV-infected men. B cells of 13 out of 14 seropositive men failed to produce Ig in response to PWM in the presence of adequate allogeneic T-helper activity. These findings suggest that HIV induces severe immunological abnormalities in T cells, B cells, and antigen-presenting cells early in infection before CD4+ T cell numbers start to decline. Impaired immunological function in subclinically HIV-infected patients may have clinical implications for vaccination strategies, in particular the use of live vaccines in groups with a high prevalence of HIV seropositivity.

Human adenovirus type 35 vector for gene therapy of brain cancer: improved transduction and bypass of pre-existing anti-vector immunity in cancer patients.

Clinical trials in malignant glioma have demonstrated excellent safety of recombinant adenovirus type 5 (Ad5) but lack of convincing efficacy. The overall low expression levels of the Coxsackie and Adenovirus receptor and the presence of high anti-Ad5-neutralizing antibody (NAb) titers in the human population are considered detrimental for consistency of clinical results. To identify an adenoviral vector better suited to infect primary glioma cells, we tested a library of fiber-chimeric Ad5-based adenoviral vectors on 12 fresh human glioma cell suspensions. Significantly improved marker gene expression was obtained with several Ad5-chimeric vectors, predominantly vectors carrying fiber molecules derived from B-group viruses (Ad11, Ad16, Ad35 and Ad50). We next tested Ad35 sero prevalence in sera derived from 90 Dutch cancer patients including 30 glioma patients and investigated the transduction efficiency of this vector in glioma cell suspensions. Our results demonstrate that the sero prevalence and the titers of NAb against Ad35 are significantly lower than against Ad5. Also, recombinant Ad35 has significantly increased ability to transfer a gene to primary glioma cells compared to Ad5. We thus conclude that Ad35 represents an interesting candidate vector for gene therapy of malignant glioma.

[The molecular epidemiology of HIV-1 in Belarus in (1996-2004): a predominance of subtype A variants and circulation of subtype B variants].

To study the molecular epidemiology of HIV-1 in Belarus, the genetic sequences of HIV-1 variants were obtained from 50 infected persons, which represented the main stages, risk groups, and geographic areas of the epidemic. The env and gag sequences were studied for HIV-1 variants from 31 persons, the env sequences were for HIV-1 variants from 18 persons, and the gag sequence was for HIV-1 variant from 1 person. Phylogenetic analysis indicated that the sequences of HIV-1 variants from 46 persons were homogenic and evolutionally closely related to IDU-A strains specific for other epidemics in the former Soviet Union are dominating in the epidemic in Belarus. Circulation of epidemiologically unrelated subtype B viruses was also established.

Is there a difference in the efficacy of peripartum antiretroviral regimens in reducing mother-to-child transmission of HIV in Africa?

BACKGROUND: Peripartum antiretroviral regimens have been shown to prevent mother-to-child transmission of HIV (MTCT) in randomized clinical trials; however, direct comparison of published results is impossible given methodological and population differences. OBJECTIVE: To directly compare the efficacy of different antiretroviral regimens in reducing the risk of 6-week MTCT rate in African breastfeeding populations. METHODS: Pooled analysis including all mother-infant pairs from any relevant trial: West African ZDV-placebo trials, Petra ZDV+3TC [two regimens A (pre/intra/post-partum) and B (intra/post-partum), placebo from Uganda and Tanzania], SAINT (NVP and Petra arm B), HIVNET012 (NVP, ultra short ZDV pp) and the Vitamin A trial (as placebo arm in South Africa). Peripartum HIV infection was any positive RNA or DNA polymerase chain reaction test < day 60. The MTCT risk was estimated at 6 weeks for each treatment arm and compared with placebo or single-dose NVP using logistic regression adjusting for maternal CD4 cell count, breastfeeding and birthweight. RESULTS: Overall, 4125 singleton live-births were included; 3629 (88%) were assessed for HIV status at 6 weeks of age. In comparison with placebo, zidovudine + lamivudine (ZDV+3TC) arm A [adjusted odds ratio (AOR), 0.23; P < 0.0001], ZDV+3TC arm B (AOR, 0.49; P < 0.001), antenatal ZDV short (AOR, 0.55; P = 0.006) and nevirapine (NVP) (AOR, 0.60; P = 0.0007) significantly reduced MTCT. In comparison with NVP, only the longest regimen of ZDV+3TC (AOR, 0.39, P < 0.0005) was significantly more effective. CONCLUSION: These results are in line with current World Health Organisation guidelines suggesting equivalence of choice between single-dose NVP and short-course ZDV, and confirm the greater efficacy of ZDV+3TC than with any single antiretroviral drug.

Baboon endogenous virus evolution and ecology.

Cross-species transmission of retroviruses among primates has recently been recognized as the source of the current epidemics of HIV-1, HIV-2 and human T cell leukemia virus type 1 (HTLV-1). The distribution of baboon endogenous virus among non-human primates resembles that of exogenous viruses and appears to be a consequence of different primate species sharing the same habitat.

Persistence of human immunodeficiency virus antigenemia in patients with the acquired immunodeficiency syndrome treated with a reverse transcriptase inhibitor, suramin. Ten-patient case-control study.

Ten homosexual men with the acquired immunodeficiency syndrome were included in a serologic follow-up study (duration, 40 weeks) of human immunodeficiency virus (HIV) antigenemia. Five of these men were treated with the reverse transcriptase inhibitor, suramin, for a period of 19 to 37 weeks. In contrast with reported changes in HIV antigen levels after treatment with zidovudine, HIV antigenemia persisted in the suramin-treated group, as well as in the untreated group. No clinical or immunologic improvement was seen in either group within the observation period. These data add evidence to the notion that monitoring HIV antigen levels helps to assess the efficacy of antiviral therapy.

Increasing genotypic and phenotypic selection from the original genomic RNA populations of HIV-1 strains LAI and MN (NM) by peripheral blood mononuclear cell culture, B-cell-line propagation and T-cell-line adaptation.

OBJECTIVE: To address the question of whether T-cell-line adaptation of the original LAI and MN (NM) HIV-1 populations biased the interpretation of the intraindividual and population-wide virus distributions. PATIENTS AND METHODS: HIV-1 genomic RNA coding for the gp120 C2V3 region was obtained from serum samples of patients LAI and MN and compared to the proviral DNA derived from simultaneously sampled peripheral blood mononuclear cells (PBMC) as well as B-and T-cell lines. RESULTS: Two (10%) of 20 clones of HIV-1 LAI RNA and none of 16 clones of the HIV-1 MN RNA carried syncytium-inducing (SI)-determining amino-acid changes. HIV-1 LAI RNA formed on SI and two non-SI (NSI) phylogenetic clusters. The HIV-1 LAI DNA in PBMC included both SI and NSI clones but lacked one NSI cluster and contributed NSI clones to the SI cluster as well as an SI clone to the NSI cluster, indicating the existence of an intermediate SI/NSI genotype. In vitro culture using either primary cells or B-cell lines yielded only SI clones, distributed over two SI/NSI mixed clusters. Long-term propagation in T-cell lines further restricted the clonality and yielded SI clones belonging to only one cluster. On the population level, HIV-1 LAI, MN and BRU sequences all clustered according to the individual host and apart from each other and separate from the epidemiological controls without notable influence of SI/NSI distinction or cell-culture adaptation. CONCLUSION: Our data demonstrate a selection bias during the cell-line adaptation of HIV-1 strains LAI and MN with more impact on phenotypic than on genotypic characteristics.

Neutralizing antibodies are positively associated with CD4+ T-cell counts and T-cell function in long-term AIDS-free infection.

OBJECTIVES: To assess the dynamics of neutralizing antibodies (NAb) in long-term AIDS-free HIV-1-infected subjects and establish correlations with known markers of disease progression. DESIGN: Cross-sectional study using sera collected from long-term non-progressors (LTNP) 8 years after seroconversion or study entry. Longitudinal study using sera collected from LTNP at 0, 0.5, 1, 2, 4, 6, 8 and 10 years after seroconversion and, as controls, from rapid progressors. METHODS: Individuals with documented AIDS-free HIV-1 infection for at least 8 years were evaluated for NAb against five heterologous HIV-1 primary isolates. In the cross-sectional study, serum viral RNA levels, CD4+ T-cell numbers and T-cell function were determined on samples collected during the eighth year of follow-up. For the longitudinal study, NAb were assessed in sequential sera taken from LTNP and rapid progressors. RESULTS: Serum neutralization titres found in individual sera differed from one HIV-1 isolate to another, were detected in 49-76% of LTNP, without correlation with the coreceptor usage of the isolate, and were positively associated with CD4+ T-lymphocyte counts (P = 0.0041) and T-cell function (P = 0.04). No correlation was found between NAb and the level of viral RNA in serum or the rate of CD4+ T-cell decline. Longitudinal analysis of sera from LTNP and rapid progressors showed that although several subjects in both groups had neutralizing activity at seroconversion, it thereafter became lower or no longer detectable. NAb were again found 1-4 years later and stably persisted in LTNP, but remained undetectable or at low levels in rapid progressors. CONCLUSIONS: NAb were preferentially found in subjects with relatively preserved T-cell function and CD4+ T-cell numbers. In these individuals, neutralizing activity against heterologous isolates increased with time. These data suggest that the capacity to produce broadly NAb is a function of the integrity of the immune system.

Qualitative and quantitative detection of HIV-1 RNA by nucleic acid sequence-based amplification.

AIM: To develop a method to detect HIV-1 viral RNA by amplifying a specific nucleic acid sequence. METHOD: The nucleic acid sequence-based amplification (NASBA) method uses the simultaneous activity of avian myeloblastosis virus reverse transcriptase, T7 RNA polymerase and RNase H to amplify a specific nucleic acid target sequence. VALIDATION: An in vitro cultured HIV-1 stock solution was used to validate the NASBA method and determine the variation in RNA measurement. CONCLUSION: Although NASBA is theoretically capable of specific amplification of RNA or DNA, it is most suitable for amplification of RNA, and therefore for detection of HIV-1 viral RNA.

Characterization of HIV-1 neutralization escape mutants.

Infection by molecularly cloned HIV-1, in the presence of a high-titre neutralizing monoclonal antibody (MAb), resulted in the selection of plaques in MT4 cells releasing HIV resistant to neutralization by the same MAb. The epitope recognized by the MAb was mapped to the V3 neutralization epitope at amino acids 305-321. The HIV-1 variants showed a reduced binding capacity for the selecting MAb as determined by immunofluorescence. Polymerase chain reaction (PCR) amplification of complementary DNA derived from viral RNA, cloning and sequencing identified a base pair (bp) change C----G at position 6663 in variant 110.5/1, predicting a change at amino acid 308 Arg----Gly. No other changes in the epitope were observed by sequencing three other variants. Differential hybridization of PCR amplified viral RNA and DNA, with oligonucleotides specific for the observed bp change or the 'wild type' sequence, indicated that the variants 110.5/1 and 110.5/7 were genotypically mixed for 308Gly/Arg. Subsequent screening of biologically 'recloned' variants 110.5/1 and 110.5/7 identified two subclones homozygous for the 308Gly change. The Arg----Gly change appears to affect the binding of the antibody to the epitope, since the linear peptide substituting 308Gly for 'wild type' 308Arg was 100 times less potent in blocking the neutralization of parental HIV. Amino-acid residue 308 thus appears to be crucial for antibody binding to the epitope. In addition, mutations distant from the monoclonal antibody binding site may also affect neutralization by antibodies recognizing the V3 loop.

Modulation of cell surface molecules during HIV-1 infection of H9 cells. An immunoelectron microscopic study.

OBJECTIVE: To study cell surface molecules and HIV-1 proteins on H9 cells 2 days after infection by immunogold electron microscopy, either in single or in double labelling using combinations of host cell-derived molecules and HIV-1 proteins. DESIGN AND METHODS: The presence of host cell antigens CD3, CD4 and human leukocyte antigen-DR (HLA-DR) and HIV-1 antigens gag p15, p17, p24 and env gp41 was evaluated using immunocytochemistry at the light microscopic level. H9 cells 2 days after infection were processed for conventional transmission electron microscopy and cryo-ultramicrotomy. Leukocyte antigens investigated were CD2, CD3, CD4 (two antibodies), CD5, CD8, CD25, CD30, CD63 antigens and HLA-DR; HIV-1-encoded antigens were gag p24, pol reverse transcriptase, and env gp41 and gp120. Double immunogold labelling was performed using reagents with different sized gold particles. For leukocyte markers, the labelling density of the cell membrane was assessed quantitatively on uninfected and infected H9 cells. RESULTS: Infected cells revealed the presence of gag p24, pol, and env gp41 and gp120 antigens on HIV-1 virions. Uninfected H9 cells showed a random distribution of cell surface molecules, including CD4 antigen, along the plasma membrane. The CD63 antigen, a lysosomal membrane glycoprotein, was located mainly in the cytoplasm of uninfected cells. Cells 2 days after infection showed CD4 labelling on sites where virions were budding from or attached to the cell surface and on free virions. Virions also showed labelling by CD3, CD5, CD25, CD30 and CD63 antibodies and anti-HLA-DR. Compared with uninfected cells, a significantly lower density was found on infected cells in labelling for CD4, CD5 and anti-HLA-DR. A significantly higher density on cells 2 days after infection was seen in CD63 labelling. CONCLUSION: During the first phase of infection host cell molecules concentrate on budding structures and newly generated HIV-1 virions. This phenomenon might contribute to the disappearance of these molecules (like the CD4 molecule) from the cell membrane after infection.

Development of HIV-1 group-specific neutralizing antibodies after seroconversion.

OBJECTIVE: To study the induction of group-specific (gs) neutralizing antibodies to HIV-1 after seroconversion. DESIGN AND METHODS: Serum samples taken sequentially from seven Dutch homosexual men and four British haemophiliacs (anonymous sample, therefore sex not known) before and after seroconversion were tested for neutralizing antibodies effective against five diverse HIV-1 strains. Strains of HIV-1 tested included isolates from the United States, Europe and Africa. RESULTS: The gs neutralizing antibody response varied between individuals. Only five of the 11 individuals studied produced detectable neutralizing antibodies to laboratory-adapted HIV-1 strains (for example, IIIB) within 32 weeks of seroconversion. Most individuals initially produced antibodies effective against US/European isolates; the response then generally broadened to include the more diverse strains, i.e., African. CONCLUSIONS: These results suggest that the gs neutralizing target for HIV-1 is poorly immunogenic in vivo and is probably not highly conserved among diverse HIV-1 strains.

Antibodies to soluble CD4 in HIV-1-infected individuals.

A direct enzyme immunoassay (EIA) using the recombinant soluble form of CD4 (sCD4) produced in rodent cells as antigen was applied to detect antibodies to CD4 in sera from HIV-1- and HIV-2-infected patients. High titers of antibodies to sCD4 were found in sera from 12.6% of the HIV-1-infected persons included in this study, but not in 120 normal human sera. The reactivity of these antibodies with sCD4 was confirmed by a Western blot analysis. A possible anti-idiotypic origin of those antibodies was thought to be unlikely in view of the lack of inhibition of the binding of the biotin-labeled monoclonal antibody (mAb) anti-Leu3a by sCD4 positive sera. Attempts to correlate the evolution of the disease with the presence or absence of antibodies to sCD4 in a panel of well documented HIV-1-seropositive cases did not reveal any clear correlation. Sera from HIV-2-infected people (nine sera analysed), sera from HIV-1-infected chimpanzees (10 sera analysed) and sera from humans immunized with a recombinant vaccinia virus expressing gp160 (10 sera analysed) scored negative for antibodies to sCD4. The possible origin and biological significance of the observed antibodies to sCD4 are discussed.

Diagnostic value of specific IgM antibodies in primary HIV infection.

Sequential serum samples from 55 homosexual men with primary HIV infection were tested for IgM anti-HIV. An early IgM response was found in 27 out of 55 (49%). In five cases IgM anti-HIV was detected 1-3 1/4 months prior to IgG anti-HIV seroconversion, as detected by a commercially available ELISA, but in no case was IgM detected prior to IgG anti-HIV seroconversion, as detected by the more sensitive GACRIA (IgG antibody captive radio-immunoassay, see Subjects and methods) and immunoblot assays. In 22 out of 23 men (96%) the primary IgM response did not persist beyond 3 months. HIV antigenaemia was found before HIV antibody seroconversion in 6 out of 55 (11%) and concomitant with HIV antibody seroconversion in 8 out of 55 (15%) subjects. A 'flu-like' illness that might be ascribed to primary HIV infection was found in 37 out of 50 men (74%). A blood sample was taken from 11 men during or within 2 weeks of the illness: no serological markers of HIV infection were detected in four subjects, HIV antigen, IgM and IgG anti-HIV were detected in another four, HIV antigen was the only marker of HIV infection in two subjects, and in one subject, IgM and IgG anti-HIV were detected but not HIV antigen. These results indicate that no conclusive value can be attached to a negative IgM test in suspected primary HIV infection, and that any role for IgM anti-HIV testing in blood donor screening is highly questionable.