Epidemiological changes of acute/recent human immunodeficiency virus type 1 infection in Barcelona, Spain (1997-2015): a prospective cohort study.

OBJECTIVE: To describe the epidemiology of acute/recent human immunodeficiency virus (HIV) infection over two decades in Barcelona (Spain). METHODS: Prospective, single-centre cohort including all patients with an acute/recent HIV infection (<180 days) since 1997. Patients were stratified into four periods. Phylogenetic analysis was performed to determine clusters of transmission. RESULTS: A total of 346 consecutive acute/recently infected patients were included. The annual proportion of recent infections among total new HIV diagnoses increased over time from 1% (29 out of 1964) to 8% (112 out of 1474) (p <0.001). Proportion of men who have sex with men (MSM) in the cohort increased from 62% (18 out of 29) to 89% (100 out of 112) (p <0.001). The proportion of migrants showed a non-significant increasing trend (24% (7 of 29) to 40% (45 of 112)) likewise the non-B subtype (0% to 22% (22 of 112)). The mean time from infection to diagnosis was 53.6 days (interquartile range (IQR) 50-57), comparable among all periods. Mean time from infection to treatment decreased over the years from 575 (IQR 467-683) to 471 (IQR 394-549) days (p <0.001) without significant differences between migrants and non-migrants (133 (IQR 71-411) versus 208 (IQR 90-523) days p 0.089). Almost 50% (152 of 311) of recently infected individuals were included in a cluster of transmission, and 92% (137 of 149) of them were MSM. CONCLUSION: The MSM population has progressively grown within acutely/recently infected patients in Barcelona, and is frequently involved in transmission clusters. Although the time between diagnosis and treatment has been reduced, the time between infection and diagnosis still needs to be shortened.

Broadly cross-neutralizing antibodies in HIV-1 patients with undetectable viremia.

Several recent studies have identified HIV-infected patients able to produce a broad neutralizing response, and the detailed analyses of their sera have provided valuable information to improve future vaccine design. All these studies have excluded patients on antiretroviral treatment and with undetectable viral loads, who have an improved B cell profile compared to untreated patients. To better understand the induction of neutralizing antibodies in patients on antiretroviral treatment with undetectable viremia, we have screened 508 serum samples from 364 patients (173 treated and 191 untreated) for a broadly neutralizing antibody (bNAb) response using a new strategy based on the use of recombinant viruses. Sera able to neutralize a minipanel of 6 recombinant viruses, including envelopes from 5 different subtypes, were found in both groups. After IgG purification, we were able to confirm the presence of IgG-associated broadly neutralizing activity in 3.7% (7 of 191) of untreated patients with detectable viremia and 1.7% (3 of 174) of aviremic patients receiving antiretroviral treatment. We thus confirm the possibility of induction of a broad IgG-associated neutralizing response in patients on antiretroviral treatment, despite having undetectable viremia. This observation is in stark contrast to the data obtained from long-term nonprogressors, whose little neutralizing activity has been attributed to the low levels of viral replication.

Ellagitannins from Tuberaria lignosa as entry inhibitors of HIV.

Screening of plants from the Iberian Peninsula for anti-human immunodeficiency virus (-HIV) activity revealed that aqueous extract of Tuberaria lignosa gave positive results. Following an activity-guided procedure, the crude extract was counterextracted, and the subsequent fractions obtained tested for their anti-HIV activity in vitro. The bioassay-guided fractionation of the extract afforded an ellagitannin enriched fraction (EEF) isolated for the first time from this species. This EEF exhibited antiviral activity against HIV in MT-2 infected cells, with an IC(50) value of 2.33mug/ml (selectivity index greater than 21). Inhibition of HIV infection by EEF appears to be mediated by CD4 down-regulation, the main receptor for HIV entry. CXCR4 and CCR5 receptors were not affected by EEF, explaining why EEF is able to inhibit R5 and X4 infections.

A new strategy based on recombinant viruses for assessing the replication capacity of HIV-1.

OBJECTIVE: In heavily pretreated patients, resistance mutations arise in both protease (PR) and reverse transcriptase (RT) sequences; however, the relative impact of PR and RT mutations on viral fitness cannot be evaluated with the majority of systems. To address this issue we have developed a model based on recombinant viruses (RVs) that allows the analysis of the replication capacity (RC) of viral populations in which PR and RT are cloned either in combination or separately. METHODS: RVs were generated for full-length polymerase (pol) gene, PR or RT sequences from nine naïve and 14 heavily pretreated HIV-infected patients in therapeutic failure. The relative RC was assessed by comparing luciferase activity between mutant RV and wild-type (wt) isolates. RESULTS: A strong decrease (>60%) in the RC of the pol RV population was observed in the 14 heavily pretreated patients as compared with the wt RVs. The analysis of PR and RT RVs from these patients showed that the decrease in RC was mainly attributable to PR sequences in three of these 14 patients and to RT sequences in seven of these patients. In the four remaining patients, PR and RT sequences independently reduced the RC of the RVs to similar extents. CONCLUSIONS: Different patterns of mutations in either PR or RT have a strong impact on RC in highly experienced HIV-infected patients.

A new strategy based on recombinant viruses as a tool for assessing drug susceptibility of human immunodeficiency virus type 1.

The emergence of drug-resistant variants during antiretroviral therapy is a serious obstacle to sustained suppression of the human immunodeficiency virus type 1 (HIV-1). For that reason, resistance assays are essential to guide clinicians in the selection of optimal treatment regimens. Genotypic assays are less expensive and results are available faster than phenotypic assays. However, in heavily experienced patients with multiple treatment failures interpretation of complex mutation patterns remains difficult, and in these cases phenotypic assays are recommended. This report describes a novel recombinant virus assay where protease (PR) and reverse transcriptase (RT) sequences derived from the plasma isolated from patients are introduced into the back-bone of an HIV molecular clone that expresses Renilla luciferase protein in the place of nef gene. All drug resistance profiles analyzed correlate with previously reported data and showed high reproducibility. This assay, in addition to a fast (completed in 10 days), precise, reproducible and automated method, presents several advantages as compared to other phenotypic assays. The system described below allows the generation of recombinant viruses with multiples cycles of replication carrying a reporter gene in their genomes. These features increase the sensitivity of the test, an important aspect to be considered in the evaluation of less fit viral isolates. In conclusion, the assay permits the quantitation of the level of resistance of clinical HIV-1 isolates to PR and RT inhibitors.

Generation of Human Monoclonal Antibodies from HIV-Infected Individuals / Generación de anticuerpos monoclonales humanos a partir de individuos infectados por VIH

Recently, the protective role of anti-HIV-1 neutralising antibodies has been «re-discovered». Few broadly protective epitopes on the HIV envelope proteins are known, which were defined by few human monoclonal antibodies (mAbs) derived many years ago from non-progressor HIV-infected individuals. It is of interest to try to generate new neutralising human mAbs both to identify new protective epitopes for vaccine design and as potential passive immunotherapy agents. In addition, it is now known that rather than recognising HIV envelope proteins, some neutralising antibodies might be autoantibodies directed to the receptor (CD4) or co-receptors (CCR5 or CXCR4) on the HIV target cells. In that context, we attempted to generate neutralising human mAbs from few HIV-1-infected individuals who, after structured antiretroviral therapy interruptions, showed good virologic and immunologic responses, including serum HIV neutralising activity. We employed the classical heterohybridoma technology and found that the best screening strategy is a primary selection of IgG-producing hybridomas, followed by secondary screenings with different antigens and assays such as HIV neutralisation activity, direct binding to HIV components, binding to HIV target cells, and to cell lines transfected with human HIV receptors and co-receptors. From a single subject, 61 IgG-producing hybridomas out of 5,760 primary wells were obtained, and preliminary data indicate the presence of six (out of 23 tested) neutralising antibodies of the X4 strain, eight anti-p24 antibodies and two antibodies that bind to the MT-2 cell line, the target of X4 strains. None of the 61 mAbs obtained reacted with cells expressing CD4, CXCR4 or CCR5. Other screening tests such as neutralisation assay with the R5 strain, binding to HIV gp120-CD4 covalent complex, and to whole chemically (aldrithiol-2)-inactivated HIV are in progress. The screening strategy is also a means to “open” the repertoire of IgG antibodies of circulating B cells in HIV-infected individuals permitting to assess the frequency of HIV-related IgG antibodies and the IgV genes encoding them, an issue largely unknown (AU)

Recientemente se ha «re-descubierto» el carácter protectivo de los anticuerpos anti-VIH neutralizantes. Se conocen sólo unos pocos epitopos protectivos en las proteínas de la envoltura del VIH definidos gracias a unos pocos anticuerpos monoclonales humanos (mAbs) generados hace ya tiempo en pacientes infectados asintomáticos. Tiene interés intentar generar otros mAbs humanos neutralizantes que puedan definir nuevos epitopos protectivos para el diseño de vacunas, y ser útiles en inmunoterapia pasiva. Además, actualmente está claro que podrían existir anticuerpos neutralizantes que en vez de ir dirigidos contra la envoltura del VIH, reconocieran al receptor (CD4) o coreceptores (CCR5 o CXCR4) en la membrana de las células diana del VIH. En este contexto, hemos intentado obtener mAbs neutralizantes a partir de aquellos pacientes VIH+ que, tras un protocolo de interrupciones estructuradas de la terapia antiretroviral, mostraron buena respuesta virológica e inmunológica, con aumento de actividad sérica neutralizante. Utilizamos la metodología de heterohybridomas convencional y hallamos que la mejor estrategia de escrutinio es la selección primaria de hibridomas secretores de IgG, y luego el escrutinio con distintos ensayos para actividad neutralizante, unión a proteínas víricas, unión a la membrana de las células diana y a células transfectadas con receptores y co-receptores del VIH. A partir de un paciente, de 5.760 microcultivos primarios, se obtuvieron 61 hibridomas productores de IgG entre los que, según datos preliminares, hay seis mAbs (de 23 probados) neutralizantes contra cepas X4, ocho anti-p24 del VIH, y dos que se unen a la componentes de la membrana de la línea MT-2, diana de las cepas X4. Ningún mAb de los 61 obtenidos se unía a CCR5, CD4 o CXCR4. Hay otros escrutinios en curso como neutralización frente a cepa R5, la unión a complejo covalente gp120-CD4 y a VIH total inactivado químicamente con aldritiol-2. El escrutinio utilizado es también un modo de abrir el repertorio de anticuerpos IgG de los linfocitos B circulantes en individuos VIH+ y averiguar la frecuencia de los anticuerpos de especificidad relacionada con el VIH y los genes IgV que los codifican, un aspecto apenas estudiado (AU)

Anti-HIV activity of medicinal plant extracts.

As part of our screening of anti-AIDS agents from natural sources, ethanolic and aqueous extracts of 15 medicinal plants widely used in the folk medicine of the Iberian Peninsula were evaluated in vitro. Most of the extracts tested were relatively nontoxic to human lymphocytic MT-2 cells, but only the extracts of Tuberaria lignosa and Sanguisorba minor magnolii exhibited anti-HIV activity in an in vitro MTT assay. The aqueous extracts of these plants showed inhibitory effects against HIV-1 induced infections in MT-2 cells at concentrations ranging from 12.5 to 50 microg/ml and 50 microg/ml, respectively. Both extracts showed no appreciable cytotoxicity at these concentrations.

Membrane raft microdomains mediate lateral assemblies required for HIV-1 infection.

HIV-1 infection triggers lateral membrane diffusion following interaction of the viral envelope with cell surface receptors. We show that these membrane changes are necessary for infection, as initial gp120-CD4 engagement leads to redistribution and clustering of membrane microdomains, enabling subsequent interaction of this complex with HIV-1 co-receptors. Disruption of cell membrane rafts by cholesterol depletion before viral exposure inhibits entry by both X4 and R5 strains of HIV-1, although viral replication in infected cells is unaffected by this treatment. This inhibitory effect is fully reversed by cholesterol replenishment of the cell membrane. These results indicate a general mechanism for HIV-1 envelope glycoprotein-mediated fusion by reorganization of membrane microdomains in the target cell, and offer new strategies for preventing HIV-1 infection.

Natural human antibodies retrieved by phage display libraries from healthy donors: polyreactivity and recognition of human immunodeficiency virus type 1gp120 epitopes.

We have characterized the human natural antibody repertoire that contains antibodies recognizing the human immunodeficiency virus type 1 (HIV-1) gp120. A panel of monovalent antigen-binding fragments (Fab) selected from IgM and IgG isotype libraries generated from peripheral blood mononuclear cells (PBMC) of a healthy, HIV-1 noninfected individual was analysed, reflecting that only IgM, but not IgG, Fab were able to recognize HIV-1 gp120. The IgM Fab antibodies were not restricted to any particular heavy chain variable region (VH) germ line gene. However, the recognition of gp120 is associated to polyreactive antibodies and all display low-affinity interaction. This correlates with the absence of any maturation process as somatic mutation or isotype switch as the nucleotide sequence analysis of the variable regions reveals they are expressed near to germline configuration. In addition, none of the antibodies showed any neutralizing activity on HIV-1-infected lymphocytes, reflecting that the natural anti-gp120 repertoire is not sufficient to neutralize HIV infection.

Antiviral activity of some South American medicinal plants.

Folk medicinal plants are potential sources of useful therapeutic compounds including some with antiviral activities. Extracts prepared from 10 South American medicinal plants (Baccharis trinervis, Baccharis teindalensis, Eupatorium articulatum, Eupatorium glutinosum, Tagetes pusilla, Neurolaena lobata, Conyza floribunda, Phytolacca bogotensis, Phytolacca rivinoides and Heisteria acuminata) were screened for in vitro antiviral activity against herpes simplex type I (HSV-1), vesicular stomatitis virus (VSV) and poliovirus type 1. The most potent inhibition was observed with an aqueous extract of B. trinervis, which inhibited HSV-1 replication by 100% at 50-200 micrograms/mL, without showing cytotoxic effects. Good activities were also found with the ethanol extract of H. acuminata and the aqueous extract of E. articulatum, which exhibited antiviral effects against both DNA and RNA viruses (HSV-1 and VSV, respectively) at 125-250 micrograms/mL. The aqueous extracts of T. pusilla (100-250 micrograms/mL), B. teindalensis (50-125 micrograms/mL) and E. glutinosum (50-125 micrograms/mL) also inhibited the replication of VSV, but none of the extracts tested had any effect on poliovirus replication.

Drastic fitness loss in human immunodeficiency virus type 1 upon serial bottleneck events.

Muller's ratchet predicts fitness losses in small populations of asexual organisms because of the irreversible accumulation of deleterious mutations and genetic drift. This effect should be enhanced if population bottlenecks intervene and fixation of mutations is not compensated by recombination. To study whether Muller's ratchet could operate in a retrovirus, 10 biological clones were derived from a human immunodeficiency virus type 1 (HIV-1) field isolate by MT-4 plaque assay. Each clone was subjected to 15 plaque-to-plaque passages. Surprisingly, genetic deterioration of viral clones was very drastic, and only 4 of the 10 initial clones were able to produce viable progeny after the serial plaque transfers. Two of the initial clones stopped forming plaques at passage 7, two others stopped at passage 13, and only four of the remaining six clones yielded infectious virus. Of these four, three displayed important fitness losses. Thus, despite virions carrying two copies of genomic RNA and the system displaying frequent recombination, HIV-1 manifested a drastic fitness loss as a result of an accentuation of Muller's ratchet effect.

In vitro selective elimination of HIV-infected cells from peripheral blood in AIDS patients by the immunotoxin DAB389CD4.

OBJECTIVES: To study the antiviral efficacy of the recombinant immunotoxin DAB389CD4 against wild-type strains of HIV and to analyse its potential toxicity in non-infected peripheral blood mononuclear cells (PBMC). DESIGN AND METHODS: PBMC from HIV-seropositive patients were cultured in the presence of DAB389CD4. After 30 days in culture, viral load was assessed by quantification of RNA levels in supernatants and HIV-specific polymerase chain reaction (PCR) was performed for measuring proviral DNA as an indicator of remaining virus in cells. To study the toxicity of DAB389CD4, PBMC from healthy donors were isolated and cell viability and lymphocyte proliferation were assessed after immunotoxin treatment. RESULTS: DAB389CD4 presented a strong antiviral activity in five of the six primary isolates decreasing p24 production in cultures to undetectable levels and eliminating selectively HIV-infected cells as measured by HIV DNA-specific PCR. One viral isolate was resistant to DAB389CD4 treatment. The immunotoxin was active against both syncytial and non-syncytial HIV strains. DAB389CD4 was not toxic in non-infected PBMC as measured by different techniques: trypan blue exclusion, methyl thiazol tetrazolium oxidation, lymphocyte proliferation, and CD4 cell count. CONCLUSIONS: DAB389CD4 showed a strong antiviral and specific activity against primary HIV isolates by killing selectively HIV-infected cells without affecting non-infected cells. This antiviral effect produced the eradication of HIV in cultures and indicated the potential use of this drug as a new therapeutic tool in combination with antiretroviral drugs. This immunotoxin would be especially interesting in the context of the marginal populations of HIV-infected cells remaining after successful antiviral treatment.

Random important alterations in HIV-1 viral quasispecies after antiviral treatment.

The evolution of HIV-1 viral populations was studied in a set of MT-2-co-cultured viruses isolated from five patients at the beginning of treatment with zidovudine and after 11-36 months of drug therapy. We first characterized the HIV-1 pol gene to detect the zidovudine-resistance mutations at codons 215 and 219. To analyse the effect that the selective pressure of zidovudine on pol exerted on other genomic regions, we also studied the env gene. The env gene sequence of virus isolated from one individual was unchanged, whereas three other sample pairs had minor alterations in env. In one individual, we detected a significant change in the env gene sequence, and so performed a clonal analysis on viruses isolated before and after treatment. In this individual, the zidovudine-resistant variant that became predominant in the resistant virus population was an undetected minority variant of the viral population before treatment was initiated. These results indicate that the evolution of quasispecies produced by selective pressure on the pol gene from zidovudine treatment could select, in a random process, important changes in other genomic regions; in particular, we describe alterations in the env gene.

Genetic analysis of herpes simplex virus type 1 isolates from recurrent lesions and clinical reinfections.

A study of the genetic variability of herpes simplex virus (HSV) type 1 from recurrent lesions and clinical reinfections was done using restriction endonuclease analysis and the RNase A mismatch cleavage method. Comparative genetic analyses of HSV-1 recurrent isolates from 1 patient and of HSV-1 isolates from different anatomic areas (vagina and lip) from another patient showed differences only in the glycoprotein B gene but not in the thymidine kinase gene even though the viruses had the same restriction endonuclease pattern. These results suggest the RNase A mismatch cleavage method is useful for epidemiologic studies of DNA viruses.

Primary genetic characterization of HIV-1 isolates from WHO-sponsored vaccine evaluation sites by the RNase-A mismatch method.

We have analyzed by the RNase-A mismatch method 35 isolates from four WHO-sponsored vaccine evaluation sites as a secondary laboratory of the WHO Network for HIV Isolation and Characterization. The application of an estimator for the establishment of genetic distances based on the RNase-A digestion patterns in combination with the phylogenetic analysis has allowed us to construct a tree with five well defined groups of viruses. Because the clustering with known reference strains, samples from Brazil could be grouped as subtype B and the majority of those from Thailand were subtype E. Some of the samples from Uganda were classified as subtype D. Isolates from Rwanda and some from Uganda were identified as subtype A viruses. These results coincide with data obtained by heteroduplex mobility assay and nucleotide sequencing in env regions. The RNase-A mismatch method combined with phylogenetic analysis permitted the primary genetic classification of 33 of 35 samples from the WHO Network.

Monensin and nigericin prevent the inhibition of host translation by poliovirus, without affecting p220 cleavage.

Addition of monensin or nigericin after poliovirus entry into HeLa cells prevents the inhibition of host protein synthesis by poliovirus. The infected cells continue to synthesize cellular proteins at control levels for at least 8 h after infection in the presence of the ionophore. Cleavage of p220 (gamma subunit of eukaryotic initiation factor 4 [eIF-4 gamma]), a component of the translation initiation factor eIF-4F, occurs to the same extent in poliovirus-infected cells whether or not they are treated with monensin. Two hours after infection there is no detectable intact p220, but the cells continue to translate cellular mRNAs for several hours at levels similar to those in uninfected cells. Nigericin or monensin prevented the arrest of host translation at all the multiplicities of poliovirus infection tested. At high multiplicities of infection, an unprecedented situation was found: cells synthesized poliovirus and cellular proteins simultaneously. Superinfection of vesicular stomatitis virus-infected HeLa cells with poliovirus led to a profound inhibition of vesicular stomatitis virus protein synthesis, while nigericin partially prevented this blockade. Drastic inhibition of translation also took place in influenza virus-infected Vero cells treated with nigericin and infected with poliovirus. These findings suggest that the translation of newly synthesized mRNAs is dependent on the integrity of p220, while ongoing cellular protein synthesis does not require an intact p220. The target of ionophore action during the poliovirus life cycle was also investigated. Addition of nigericin at any time postinfection profoundly blocked the synthesis of virus RNA, whereas viral protein synthesis was not affected if nigericin was added at 4 h postinfection. These results agree well with previous findings indicating that inhibitors of phospholipid synthesis or vesicular traffic interfere with poliovirus genome replication. Therefore, the action of nigericin on the vesicular system may affect poliovirus RNA synthesis. In conclusion, monensin and nigericin are potent inhibitors of poliovirus genome replication that prevent the shutoff of host translation by poliovirus while still permitting cleavage of p220.

Molecular epidemiology of HIV-1 in Madrid.

Thirteen HIV-1 isolates from patients of different risk groups in Madrid (Spain) have been analyzed at the genetic level. Two distinct lineages of subtype B have been detected among the HIV-1 circulating in this area: one was related to SF-2/RF strains, whereas the other consists of a more heterogeneous group related to reference strain III-B. Variants of each lineage appeared to circulate preferentially within a risk group: III-B among intravenous drug users, and RF/SF-2 among male homosexuals.

Dilute passage promotes expression of genetic and phenotypic variants of human immunodeficiency virus type 1 in cell culture.

We have studied the extent of genetic and phenotypic diversification of human immunodeficiency virus type 1 (HIV-1) upon 15 serial passages of clonal viral populations in MT-4 cell cultures. Several genetic and phenotypic modifications previously noted during evolution of HIV-1 in infected humans were also observed upon passages of the virus in cell culture. Notably, the transition from non-syncytium-inducing to syncytium-inducing phenotype (previously observed during disease progression) and fixation of amino acid substitutions at the main antigenic loop V3 of gp120 were observed in the course of replication of the virus in MT-4 cell cultures in the absence of immune selection. Interestingly, most genetic and phenotypic alterations occurred upon passage of the virus at a low multiplicity of infection (0.001 infectious particles per cell) rather than at a higher multiplicity of infection (0.1 infectious particles per cell). The degree of genetic diversification attained by HIV-1, estimated by the RNase A mismatch cleavage method and by nucleotide sequencing, is of about 0.03% of genomic sites mutated after 15 serial passages. This value is not significantly different from previous estimates for foot-and-mouth disease virus when subjected to a similar process and analysis. We conclude that several genetic and phenotypic modifications of HIV-1 previously observed in vivo occur also in the constant environment provided by a cell culture system. Dilute passage promotes in a highly significant way the expression of deviant HIV-1 genomes.