Characterization of neutralizing profiles in HIV-1 infected patients from whom the HJ16, HGN194 and HK20 mAbs were obtained.

Several new human monoclonal antibodies (mAbs) with a neutralizing potential across different subtypes have recently been described. Three mAbs, HJ16, HGN194 and HK20, were obtained from patients within the HIV-1 cohort of the Institute of Tropical Medicine (ITM). Our aim was to generate immunization antibodies equivalent to those seen in plasma. Here, we describe the selection and characterization of patient plasma and their mAbs, using a range of neutralization assays, including several peripheral blood mononuclear cell (PBMC) based assays and replicating primary viruses as well as cell line based assays and pseudoviruses (PV). The principal criterion for selection of patient plasma was the activity in an 'extended incubation phase' PBMC assay. Neutralizing Abs, derived from their memory B cells, were then selected by ELISA with envelope proteins as solid phase. MAbs were subsequently tested in a high-throughput HOS-PV assay to assess functional neutralization. The present study indicates that the strong profiles in the patients' plasma were not solely due to antibodies represented by the newly isolated mAbs. Although results from the various assays were divergent, they by and large indicate that neutralizing Abs to other epitopes of the HIV-1 envelope are present in the plasma and synergy between Abs may be important. Thus, the spectrum of the obtained mAbs does not cover the range of cross-reactivity seen in plasma in these carefully selected patients irrespective of which neutralization assay is used. Nevertheless, these mAbs are relevant for immunogen discovery because they bind to the recombinant glycoproteins to which the immune response needs to be targeted in vivo. Our observations illustrate the remaining challenges required for successful immunogen design and development.

Unravelling the antigenic landscape of the HIV-1 subtype A envelope of an individual with broad cross-neutralizing antibodies using phage display peptide libraries.

Broad cross-neutralizing antibodies from persons infected with HIV-1 target a variety of epitopes. Identification of these HIV-1 epitopes may result in an optimal panel of antigenic peptides to be included in a prophylactic vaccine. Phage display peptide libraries were used to unravel the antigenic landscape of an individual (ITM1) infected with HIV-1 subtype A with broad cross-neutralizing antibodies. A stringent selection strategy resulted in the identification of 60 unique HIV-1 peptide phage, which were subjected to sequence analysis and mapped onto the ITM1 envelope sequences. Four groups of peptide phages were found: the first group (n=11) were similar with the tip of the V3 loop (KxxHxGPxxxF); the second group (n=11) represented the gp41 principal immunodominant domain (CxGxLxCTxNxP); the third group (n=16) could be localized in the V2 loop (KxxxHxxxY); and the fourth group (n=22) mimicked a conformational epitope (Hxx(S)/(T)NxK). All but the V2-binding antibodies were conserved over the 11 years of follow-up. A neutralization inhibition assay revealed the contribution of the V3 antibodies to the neutralizing capacity of the ITM1 plasma. Overall, the ITM1 immunogenic landscape was mapped and a part of the origin of this broad cross-neutralizing activity was demonstrated.

Evolution of antibody landscape and viral envelope escape in an HIV-1 CRF02_AG infected patient with 4E10-like antibodies.

BACKGROUND: A minority of HIV-1 infected individuals develop broad cross-neutralizing (BCN) plasma antibodies that are capable of neutralizing a spectrum of virus variants belonging to different HIV-1 clades. The aim of this study was to identify the targeted epitopes of an individual with BCN plasma antibodies, referred to as ITM4, using peptide phage display. This study also aimed to use the selected mimotopes as tools to unravel the evolution of the antibody landscape and the viral envelope escape which may provide us with new insights for vaccine design. RESULTS: This study led us to identify ITM4 plasma antibodies directed to the 4E10 epitope located in the gp41 membrane-proximal external region (MPER). Analysis of antibody specificities revealed unusual immunogenic properties of the ITM4 viral envelope, as not only the V3 loop and the gp41 MPER but also the C1 and lentivirus lytic peptide 2 (LLP2) region seem to be targets of the immune system. The 4E10-like antibodies are consistently elicited during the 6-year follow up period. HIV-1 ITM4 pseudoviruses showed an increasing resistance over time to MPER monoclonal antibodies 4E10 and 2F5, although no changes are found in the critical positions of the epitope. Neutralization of COT6.15 (subtype C; 4E10-sensitive) pseudoviruses with alanine substitutions in the MPER region indicated an overlapping specificity of the 4E10 monoclonal antibody and the ITM4 follow up plasma. Moreover the 4E10-like antibodies of ITM4 contribute to the BCN capacity of the plasma. CONCLUSIONS: Using ITM4 BCN plasma and peptide phage display technology, we have identified a patient with 4E10-like BCN antibodies. Our results indicate that the elicited 4E10-like antibodies play a role in virus neutralization. The viral RNA was isolated at different time points and the ITM4 envelope sequence analysis of both early (4E10-sensitive) and late (4E10-resistant) viruses suggest that other regions in the envelope, outside the MPER region, contribute to the accessibility and sensitivity of the 4E10 epitope. Including ITM4 specific HIV-1 Env properties in vaccine strategies may be a promising approach.

HIV type 1 subtype A envelope genetic evolution in a slow progressing individual with consistent broadly neutralizing antibodies.

Studies of viruses taken from individuals with broad cross-neutralizing antibodies against primary isolates may reveal novel antibody specificities and their associated epitopes that could be useful for immunogen design. We report on the Env antigenic variability of a slow progressing HIV-1 subtype A-infected donor with consistent broad cross-neutralizing antibodies during the second decade of disease progression after vertical transmission. The Env evolution is characterized by a genetic shift to variants with altered V1-V5 loop sequences, marked by consecutive changes in V1, V4-V5, and C3 and largely conserved V2 and V3 loop sequences. Major V1 Env sequence expansion, variation by a duplication event, and cumulative addition of cysteine residues and potential N-glycosylation sites over time may contribute to escape from antibody pressure directed to Env receptor domains by changing the exposure of neutralization-sensitive epitopes. Conservation of functional epitopes may correlate with the continued presence of broad cross-neutralizing antibodies.

Human immunodeficiency virus type 1 escape from cyclotriazadisulfonamide-induced CD4-targeted entry inhibition is associated with increased neutralizing antibody susceptibility.

Continuous specific downmodulation of CD4 receptor expression in T lymphocytes by the small molecule cyclotriazadisulfonamide (CADA) selected for the CADA-resistant human immunodeficiency virus type 1 (HIV-1) NL4.3 virus containing unique mutations in the C4 and V5 regions of gp120, likely stabilizing the CD4-binding conformation. The amino acid changes in Env were associated with decreased susceptibility to anti-CD4 monoclonal antibody treatment of the cells and with higher susceptibility of the virus to soluble CD4. In addition, the acquired ability of a CADA-resistant virus to infect cells with low CD4 expression was associated with an increased susceptibility of the virus to neutralizing antibodies from sera of several HIV-1-infected patients.

Virological response to highly active antiretroviral therapy in patients infected with human immunodeficiency virus type 2 (HIV-2) and in patients dually infected with HIV-1 and HIV-2 in the Gambia and emergence of drug-resistant variants.

Drug design, antiretroviral therapy (ART), and drug resistance studies have focused almost exclusively on human immunodeficiency virus type 1 (HIV-1), resulting in limited information for patients infected with HIV-2 and for those dually infected with HIV-1 and HIV-2. In this study, 20 patients, 12 infected with HIV-2 and 8 dually infected with HIV-1 and HIV-2, all treated with zidovudine (ZDV), lamivudine (3TC), and lopinavir-ritonavir (LPV/r), were followed up longitudinally for about 3 years. For 19/20 patients, viral loads were reduced to undetectable levels; the patient whose viral load remained detectable reported adverse effects associated with LPV/r that had caused him to stop taking all the drugs. HIV-2 strains containing mutations in both the protease and the reverse transcriptase gene that may confer drug resistance were observed in two patients with viral rebound, as early as 130 days (4.3 months) after the initiation of therapy. We conclude that the combination of ZDV, 3TC, and LPV/r is able to provide efficient and durable suppression of HIV-1 and HIV-2 for as long as 3 years in HIV-2-infected and dually infected patients. However, the emergence of HIV-1 and HIV-2 strains containing drug-resistant mutations can compromise the efficacy of this highly active ART.

Presence of a multidrug-resistance mutation in an HIV-2 variant infecting a treatment-naive individual in Caio, Guinea Bissau.

We report the possible transmission of drug-resistant human immunodeficiency virus type 2. A 66-year-old woman from rural Guinea Bissau who had no obvious antiretroviral exposure was found to harbor a variant with the multidrug-resistance mutation Q151M. Finding this mutation among a drug-naive population presents an important public health issue that needs to be addressed for treatment to be effective.

Neutralization patterns and evolution of sequential HIV type 1 envelope sequences in HIV type 1 subtype B-infected drug-naive individuals.

To design a vaccine that will remain potent against HIV-1, the immunogenic regions in the viral envelope that tend to change as well as those that remain constant over time must be identified. To determine the neutralization profiles of sequential viruses over time and study whether neutralization patterns correlate with sequence evolution, 12 broadly neutralizing plasmas from HIV-1 subtype B-infected individuals were tested for their ability to neutralize sequential primary HIV-1 subtype B viruses from four individuals. Three patterns of neutralization were observed, including a loss of neutralization sensitivity by viruses over time, an increase in neutralization sensitivity by sequential viruses, or a similarity in the sensitivity of sequential viruses to neutralization. Seven to 11 gp160 clones from each sequential virus sample were sequenced and analyzed to identify mutational patterns. Analysis of the envelope sequences of the sequential viruses revealed changes characteristic of the neutralization patterns. Viruses that evolved to become resistant to neutralizing antibodies also evolved with diverse sequences, with most of the changes being due to nonsynonymous mutations occurring in the V1/V2, as well as in the constant regions (C2, C3, C4), the most changes occurring in the C3. Viruses from the patient that evolved to become more sensitive to neutralization exhibited less sequence diversity with fewer nonsynonymous changes that occurred mainly in the V1/V2 region. The V3 region remained constant over time for all the viruses tested. This study demonstrates that as viruses evolve in their host, they either become sensitive or resistant to neutralization by antibodies in heterologous plasma and mutations in different envelope regions account for these changes in their neutralization profiles.

Optimization of the oligonucleotide ligation assay, a rapid and inexpensive test for detection of HIV-1 drug resistance mutations, for non-North American variants.

OBJECTIVE: We evaluated the feasibility of the oligonucleotide ligation assay (OLA), a specific, sensitive, and economical ligase-based point mutation assay designed to detect HIV-1 drug-resistance mutations at 12 codons of HIV-1 subtype B pol, for potential use in resource-poor settings. METHODS: Specimens from HIV-1-infected individuals collected by 7 international laboratories, including subtypes A, B, C, D, F, G, J, and recombinants AE and AG, were tested by the OLA developed for HIV-1 subtype B. Common polymorphisms that interfered with reactivity of the OLA were identified and modified probes designed and evaluated. RESULTS: 92.5% (2,410) of 2,604 codons in specimens from 217 individuals were successfully genotyped by the subtype B OLA. A high rate (range 8.3%-31.2%) of indeterminate results (negative OLA reaction for both mutant and wild type) was observed for 5 codons. Modified probes at reverse transcriptase codons 151 and 184 and protease codon 90 increased the rate of valid OLA to 96.1%. CONCLUSIONS: The OLA designed for HIV-1 subtype B genotyped most pol codons in non-B subtypes from Asia and Africa but was improved by addition of several modified probes. International laboratories experienced in molecular techniques were able to perform the OLA.

Development and evaluation of an oligonucleotide ligation assay for detection of drug resistance-associated mutations in the human immunodeficiency virus type 2 pol gene.

Human immunodeficiency virus type 2 (HIV-2) is naturally resistant to several antiretroviral drugs, including all of the non-nucleoside reverse transcriptase inhibitors and the entry inhibitor T-20, and may have reduced susceptibility to some protease inhibitors. These resistance properties make treatment of HIV-2 patients difficult, with very limited treatment options. Therefore, early detection of resistance mutations is important for understanding treatment failures and guiding subsequent therapy decisions. With the Global Fund Initiative, a substantial number of HIV-2 patients in West Africa will receive antiretroviral therapy. Therefore, development of cheaper and more sustainable resistance assays, such as the oligonucleotide ligation assay (OLA), is a priority. In this study, we designed oligonucleotide probes to detect the Q151M mutation, associated with phenotypic resistance to zidovudine, didanosine, zalcitabine, and stavudine, and the M184V mutation, associated with phenotypic resistance to lamivudine and emtricitabine, in HIV-2. The assay was successfully developed and evaluated with 122 samples from The Gambia, Guinea Bissau, The Netherlands, and Sweden. The overall sensitivity of the assay was 98.8%, with 99.2% for Q151M and 98.4% for M184V. OLA results were compared with sequencing to give high concordances of 98.4% (Q151M) and 97.5% (M184V). OLA demonstrated a higher sensitivity for detection of minor variants as a mixture of wild-type and mutant viruses in cases when sequencing detected only the major population. In conclusion, we have developed a simple, easy-to-use, and economical assay for genotyping of drug resistance in HIV-2 that is more sustainable for use in resource-poor settings than is consensus sequencing.

Neutralization kinetics of sensitive and resistant subtype B primary human immunodeficiency virus type 1 isolates.

The aim of the study was to determine if sensitive and resistant human immunodeficiency virus type 1 (HIV-1) subtype B primary isolates have different neutralization kinetics. Neutralization assays were undertaken where either the time allowed for virus to react with antibodies or the subsequent period of this mixture's exposure to target cells were varied. The relative neutralization sensitivity/resistance is a reproducible property of the isolates. In a minority of combinations, the titre falls exponentially for as long as the free virions are exposed to antibody. In the remainder, neutralization kinetics shows deviations which may be attributed to events occurring after the virus-antibody mixture is added to the target cells: significant neutralization with minimal exposure of the free virions to antibody; a plot where reduction in virus titre is parallel to the duration of the incubation phase of the assay. Neutralization rate constants are similar for primary HIV-1 SF33, HIV-1 SF162, and HIV-1 89.6, reaching 5 x 10(5)-1 x 10(6)/M sec for the monoclonal antibody IgG1 b12. However, although increased antibody levels produced greater reductions in virus titre the rate of neutralization was not proportional to the antibody concentration. Neutralization of either the free virion or cell-associated virus does not correlate with the resistance/sensitivity of primary subtype B isolates. The target cells play an active role, so that in designing neutralization assays with primary isolates of HIV-1, events following the virus-antibody complex binding to the cell surface have to be taken into consideration.

Replicative fitness of CCR5-using and CXCR4-using human immunodeficiency virus type 1 biological clones.

CCR5-tropic viruses cause the vast majority of new HIV-1 infections while about half of the individuals infected with HIV-1 manifest a co-receptor switch (CCR5 (R5) to CXCR4 (X4)) prior to accelerated disease progression. The underlying biological mechanisms of X4 outgrowth in AIDS patients are still poorly understood. Although X4 viruses have been associated with increased "virulence" in vivo, in vitro replication and cytopathicity studies of X4 and R5 viruses have led to conflicting conclusions. We studied the replicative fitness of HIV-1 biological clones with different co-receptor tropism, isolated from four AIDS patients. On average, R5 and X4 clones replicated equally well in mitogen-activated T cells. In contrast, X4 variants were transferred more efficiently from dendritic cells to autologous CD4+ T cells. These observations suggest that interaction between X4 viruses, DC and T cells might contribute to the preferential outgrowth of X4 viruses in AIDS patients.

Development, evaluation, and validation of an oligonucleotide probe hybridization assay to subtype human immunodeficiency virus type 1 circulating recombinant form CRF02_AG.

We have developed and validated an oligonucleotide probe hybridization assay for human immunodeficiency virus type 1 (HIV-1) circulating recombinant form (CRF) CRF02_AG. In the p17 coding region of the gag gene, a CRF02_AG-specific signature pattern was observed. Five working probes were designed to discriminate CRF02_AG infections from infections by all other documented subtypes and CRFs in an enzyme-linked immunosorbent assay-based oligonucleotide probe hybridization assay. Nucleic acids were extracted from a panel of HIV-1-positive plasma samples from Cameroon, Bénin, Côte d'Ivoire, Kenya, Zambia, and Belgium and from blood spots from The Gambia. CRF02_AG (n = 147) and non-CRF02 (n = 100) samples were analyzed to evaluate and validate the oligonucleotide probe hybridization assay. The CRF02_AG-specific oligonucleotide probe hybridization assay has a high sensitivity and specificity, with good positive and negative predictive values in regions of high and low prevalence. A validation of the assay with West and West Central African samples indicated a sensitivity of 98.4% and a specificity of 96.7%. The oligonucleotide probe hybridization assay as a diagnostic tool will allow for rapid screening for CRF02_AG. This could be used to track the HIV epidemic in terms of documenting the real prevalence of CRF02_AG strains and will complement efforts in vaccine development. Moreover, this technology can easily be applied in laboratories in developing countries.

Comparison of predicted scaffold-compatible sequence variation in the triple-hairpin structure of human imunodeficiency virus type 1 gp41 with patient data.

It has been proposed that the ectodomain of human immunodeficiency virus type 1 (HIV-1) gp41 (e-gp41), involved in HIV entry into the target cell, exists in at least two conformations, a pre-hairpin intermediate and a fusion-active hairpin structure. To obtain more information on the structure-sequence relationship in e-gp41, we performed in silico a full single-amino-acid substitution analysis, resulting in a Fold Compatible Database (FCD) for each conformation. The FCD contains for each residue position in a given protein a list of values assessing the energetic compatibility (ECO) of each of the 20 natural amino acids at that position. Our results suggest that FCD predictions are in good agreement with the sequence variation observed for well-validated e-gp41 sequences. The data show that at a minECO threshold value of 5 kcal/mol, about 90% of the observed patient sequence variation is encompassed by the FCD predictions. Some inconsistent FCD predictions at N-helix positions packing against residues of the C helix suggest that packing of both peptides may involve some flexibility and may be attributed to an altered orientation of the C-helical domain versus the N-helical region. The permissiveness of sequence variation in the C helices is in agreement with FCD predictions. Comparison of N-core and triple-hairpin FCDs suggests that the N helices may impose more constraints on sequence variation than the C helices. Although the observed sequences of e-gp41 contain many multiple mutations, our method, which is based on single-point mutations, can predict the natural sequence variability of e-gp41 very well.