Amparo Álvarez

Negatively Charged Glyconanoparticles Modulate and Stabilize the Secondary Structures of a gp120 V3 Loop Peptide: Toward Fully Synthetic HIV Vaccine Candidates

The third variable region (V3 peptide) of the HIV-1 gp120 is a major immunogenic domain of HIV-1. Controlling the formation of the immunologically active conformation is a crucial step to the rational design of fully synthetic candidate vaccines. Herein, we present the modulation and stabilization of either the α-helix or β-strand conformation of the V3 peptide by conjugation to negatively charged gold glyconanoparticles (GNPs). The formation of the secondary structure can be triggered by the variation of the buffer concentration and/or pH as indicated by circular dichoism. The peptide on the GNPs shows increased stability toward peptidase degradation as compared to the free peptide. Moreover, only the V3β-GNPs bind to the anti-V3 human broadly neutralizing mAb 447-52D as demonstrated by surface plasmon resonance (SPR). The strong binding of V3β-GNPs to the 447-52D mAb was the starting point to address its study as immunogen. V3β-GNPs elicit antibodies in rabbits that recognize a recombinant gp120 and the serum displayed low but consistent neutralizing activity. These results open up the way for the design of new fully synthetic HIV vaccine candidates.

High prevalence of HIV-1 subtype G and natural polymorphisms at the protease gene among HIV-infected immigrants in Madrid.

Objectives Genetic characterization of HIV-1 subtypes among immigrants and natives infected overseas. Methods Phylogenetic analysis of HIV-1 protease sequences obtained from 109 foreigners (mainly Africans) and 32 native individuals infected overseas attending a reference HIV/AIDS centre located in Madrid, Spain. Results The overall rate of infection with HIV-1 non-B subtypes was 50.3% (71/141). Whereas 94.3% (67/71) belonged to immigrants (mostly Africans, 60/67), only 5.6% (4/71) were from native individuals (P < 0.05). The distribution of non-B subtypes was: 49 G, eight C, six A, four D, two F and two H. The high prevalence of subtype G was mainly related to individuals from west-central Africa. Interestingly, substitutions at three or more positions associated with protease inhibitor (PI) resistance were recognized in 52.6% of naive subjects carrying non-B subtypes, but only in 8% of those infected with B viruses (P < 0.05). The genotypes most frequently recognized among non-B and B subtypes occurred, respectively, at positions 36 (100 versus 12%), 20 (77.2 versus 0%), 63 (40.3 versus 64%), 82 (17.5 versus 0%), 10 (14 versus 12%), 77 (3.5 versus 34%), and 71 (0 versus 2%). Accordingly, changes I-36 and I-20 may be considered specific genetic markers for non-B, group M variants and subtype G infections, respectively. Conclusion Nearly two-thirds of foreigners with HIV-1 infection in Madrid carry non-B subtypes, subtype G (protease) being the most common among west-central African immigrants. The high rate of natural polymorphisms at the protease gene in non-B viruses may compromise the response to PI. Therefore, HIV subtyping should be considered in treatment guidelines.

Safety and immunogenicity of a modified pox vector-based HIV/AIDS vaccine candidate expressing Env, Gag, Pol and Nef proteins of HIV-1 subtype B (MVA-B) in healthy HIV-1-uninfected volunteers: A phase I clinical trial (RISVAC02).

BACKGROUND To investigate the safety and immunogenicity of a modified vaccinia virus Ankara vector expressing HIV-1 antigens from clade B (MVA-B), a phase-I, doubled-blind placebo-controlled trial was performed. METHODS 30 HIV-uninfected volunteers at low risk of HIV-1 infection were randomly allocated to receive 3 intramuscular injections (1×10(8)pfu/dose) of MVA-B (n=24) or placebo (n=6) at weeks 0, 4 and 16. All volunteers were followed 48 weeks. Primary end-points were adverse events and immunogenicity. RESULTS A total of 169 adverse events were reported, 164 of grade 1-2, and 5 of grade 3 (none related to vaccination). Overall 75% of the volunteers showed positive ELISPOT responses at any time point. The magnitude (median) of the total responses induced was 288SFC/10(6)PBMC at week 18. Antibody responses against Env were observed in 95% and 72% of vaccinees at week 18 and 48, respectively. HIV-1 neutralizing antibodies were detected in 33% of volunteers. CONCLUSIONS MVA-B was safe, well tolerated and elicited strong and durable T-cell and antibody responses in 75% and 95% of volunteers, respectively. These data support further exploration of MVA-B as an HIV-1 vaccine candidate. Clinical Trials.gov identifier: NCT00679497.

SJ23B, a jatrophane diterpene activates classical PKCs and displays strong activity against HIV in vitro

Existence of virus reservoirs makes the eradication of HIV infection extremely difficult. Current drug therapies neither eliminate these viral reservoirs nor prevent their formation. Consequently, new strategies are needed to target these reservoirs with the aim of decreasing their size. We analysed a series of jatrophane diterpenes isolated from Euphorbia hyberna and we found that one of them, SJ23B, induces the internalization of the HIV-1 receptors CD4, CXCR4 and CCR5 and prevents R5 and X4 viral infection in human primary T cells at the nanomolar range. Moreover, SJ23B is a potent antagonist of HIV-1 latency. Using Jurkat-LAT-GFP cells, a model for HIV-1 latency, we found that prostratin and SJ23B activate HIV-1 gene expression, with SJ23B being at least 10-fold more potent than prostratin. SJ23B did not elicit transforming foci activity in NIH 3T3 cells but is a potent activator of PKCalpha and delta as measured by in vitro kinase assays and by cellular translocation experiments. By using isoform-specific PKC inhibitors we found that cPKCs are critical for SJ23B-induced HIV-1 reactivation. We also showed that both SJ23B-induced IkappaBalpha degradation and NF-kappaB activation were inhibited by the classical PKC inhibitor, Gö6976. Accordingly, SJ23B synergizes with ionomycin to translocate PKCalpha to the plasma membrane and to activate the NF-kappaB pathway. Moreover, SJ23B activates both NF-kappaB and Sp1-dependent transcriptional activities in primary T cells. We have shown that diterpene jatrophanes represent a new member of anti-AIDS agents that could be developed for mitigating HIV reactivation.

A sensitive phenotypic assay for the determination of human immunodeficiency virus type 1 tropism

OBJECTIVES To develop a sensitive phenotypic assay based on recombinant viruses (RVs) for characterizing HIV-1 tropism. METHODS Viral tropism was assessed in 159 plasma samples. The env gene was amplified and ligated into pNL-lacZ/env-Ren, which carries a luciferase reporter gene. Resulting constructs were transfected into HEK293T cells to generate RVs. To assess co-receptor tropism, U87.CD4.CXCR4/CCR5 cells were infected and luciferase activity was measured. RESULTS RVs containing env from different HIV-1 subtypes were replication competent. Minor variants were detectable in 1% of the viral population. Tropism was determined in 65% of samples with a viral load of <1000 copies/mL. The phenotypic assay described here was validated with the Trofile™ and Trofile™ES assays. Considering the Trofile™ assay as a reference, the sensitivity for R5 and R5X4/X4 detection was 90% and 77%, and the specificity was 77% and 90%, respectively. Our assay was 86% concordant with Trofile™ (90% for R5 and 77% for R5X4/X4). When our system was compared with Trofile™ES, the concordance was 89% (86% for R5 and 92% for R5X4/X4), the sensitivity for R5 was 86% and for R5X4/X4 was 92%, and the specificity for R5 was 92% and for R5X4/X4 was 86%. The phenotypic results were compared with those obtained using the following V3 genotypic prediction tools: position-specific scoring matrix; geno2pheno[coreceptor]; C4.5; C4.5 using positions 8 and 12; PART; support vector machines; and the charge rule. CONCLUSIONS We describe a system to assess co-receptor tropism based on the generation of chimeric replication-competent viruses with high sensitivity in the detection of minor populations. A good correlation of our results with Trofile™ assays was found.

Prevalence of Human Immunodeficiency Virus Type 1 (HIV-1) Non-B Subtypes in Foreigners Living in Madrid, Spain, and Comparison of the Performances of the AMPLICOR HIV-1 MONITOR Version 1.0 and the New Automated Version 1.5

ABSTRACT Plasma specimens collected in 1999 from 32 human immunodeficiency virus type 1 (HIV-1)-infected foreigners living in Madrid, Spain, were examined for the presence of non-B subtypes. Furthermore, plasma viremia was quantified using two different AMPLICOR HIV-1 MONITOR tests, version 1.0 and the new upgraded and automated version 1.5 (COBAS). Most patients came from Africa, where they most likely had acquired HIV-1 infection through sexual contact. HIV-1 genetic subtyping was based on the phylogenetic analysis of theprotease gene. Twenty-two subtype B, six subtype G, two subtype C, one subtype A, and one D subtype infection were found. Overall, non-B subtypes represented 31.25% of the study population. Irrespective of the HIV-1 variant, viral load values above the detection limit (200 HIV RNA copies/ml) increased from 56.2 to 71.9% for results obtained using MONITOR version 1.0 and COBAS, respectively. Moreover, significant differences in viral load values (>0.5 logs) were recognized in up to 37.5% of samples. In summary, COBAS seemed to be more reliable for testing plasma viral load in HIV-infected immigrants living in Spain, one third of whom carried non-B subtypes.

Broadly Cross-Neutralizing Antibodies in HIV-1 Patients with Undetectable Viremia

ABSTRACT 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.

ISG15 Is Upregulated in Respiratory Syncytial Virus Infection and Reduces Virus Growth through Protein ISGylation

ABSTRACT Human respiratory syncytial virus (RSV), for which neither a vaccine nor an effective therapeutic treatment is currently available, is the leading cause of severe lower respiratory tract infections in children. Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein that is highly increased during viral infections and has been reported to have an antiviral or a proviral activity, depending on the virus. Previous studies from our laboratory demonstrated strong ISG15 upregulation during RSV infection in vitro. In this study, an in-depth analysis of the role of ISG15 in RSV infection is presented. ISG15 overexpression and small interfering RNA (siRNA)-silencing experiments, along with ISG15 knockout (ISG15−/−) cells, revealed an anti-RSV effect of the molecule. Conjugation inhibition assays demonstrated that ISG15 exerts its antiviral activity via protein ISGylation. This antiviral activity requires high levels of ISG15 to be present in the cells before RSV infection. Finally, ISG15 is also upregulated in human respiratory pseudostratified epithelia and in nasopharyngeal washes from infants infected with RSV, pointing to a possible antiviral role of the molecule in vivo. These results advance our understanding of the innate immune response elicited by RSV and open new possibilities to control infections by the virus. IMPORTANCE At present, no vaccine or effective treatment for human respiratory syncytial virus (RSV) is available. This study shows that interferon-stimulated gene 15 (ISG15) lowers RSV growth through protein ISGylation. In addition, ISG15 accumulation highly correlates with the RSV load in nasopharyngeal washes from children, indicating that ISG15 may also have an antiviral role in vivo. These results improve our understanding of the innate immune response to RSV and identify ISG15 as a potential target for virus control.

Broadly Neutralizing Antibodies and their Significance for HIV-1 Vaccines

Despite extensive efforts, a preventive HIV vaccine has not yet been obtained and remains the main challenge in the field of AIDS research. Empirical approaches which have proved successful for many vaccines are not sufficient to tackle HIV-1 and new strategies to design effective preventive AIDS vaccines are critical. To this aim, further understanding of the mechanisms of action of neutralizing antibodies is required. In this review we summarize our current knowledge on the structure of the gp160 viral envelope and the dynamics of viral entry, the evolution of humoral response in HIV-infected patients and the mechanisms of viral escape. Finally, we describe the few neutralizing antibodies with activity against a broad spectrum of circulating HIV strains and their relevance in the design of new candidates to HIV-1 vaccines.

HIV-1 Subtype J Recombinant Viruses in Spain

Genetic characterization of HIV-1 was carried out in two women who originated in Cameroon and Equatorial Guinea, respectively, and who were diagnosed more recently as HIV-1 seropositive in Madrid, Spain. Phylogenetic studies showed that the protease-encoding gene from both individuals clustered with subtype J sequences with a high bootstrap. However, env sequences clustered with subtypes A and C, respectively. This work represents the first characterization of HIV-1 containing subtype J-like genomic regions in Spain.