Photoimmunotherapy Using Cationic and Anionic Photosensitizer-Antibody Conjugates against HIV Env-Expressing Cells.

Different therapeutic strategies have been investigated to target and eliminate HIV-1-infected cells by using armed antibodies specific to viral proteins, with varying degrees of success. Herein, we propose a new strategy by combining photodynamic therapy (PDT) with HIV Env-targeted immunotherapy, and refer to it as HIV photoimmunotherapy (PIT). A human anti-gp41 antibody (7B2) was conjugated to two photosensitizers (PSs) with different charges through different linking strategies; "Click" conjugation by using an azide-bearing porphyrin attached via a disulfide bridge linker with a drug-to-antibody ratio (DAR) of exactly 4, and "Lysine" conjugation by using phthalocyanine IRDye 700DX dye with average DARs of 2.1, 3.0 and 4.4. These photo-immunoconjugates (PICs) were compared via biochemical and immunological characterizations regarding the dosimetry, solubility, and cell targeting. Photo-induced cytotoxicity of the PICs were compared using assays for apoptosis, reactive oxygen species (ROS), photo-cytotoxicity, and confocal microscopy. Targeted phototoxicity seems to be primarily dependent on the binding of PS-antibody to the HIV antigen on the cell membrane, whilst being independent of the PS type. This is the first report of the application of PIT for HIV immunotherapy by killing HIV Env-expressing cells.

A recombinant gp145 Env glycoprotein from HIV-1 expressed in two different cell lines: Effects on glycosylation and antigenicity.

The envelope glycoprotein (Env) of the human immunodeficiency virus (HIV), has been the primary target for the development of a protective vaccine against infection. The extensive N-linked glycosylation on Env is an important consideration as it may affect efficacy, stability, and expression yields. The expression host has been shown to influence the extent and type of glycosylation that decorates the protein target. Here, we report the glycosylation profile of the candidate subtype C immunogen CO6980v0c22 gp145, which is currently in Phase I clinical trials, produced in two different host cells: CHO-K1 and Expi293F. The amino acid sequence for both glycoproteins was confirmed to be identical by peptide mass fingerprinting. However, the isoelectric point of the proteins differed; 4.5-5.5 and 6.0-7.0 for gp145 produced in CHO-K1 and Expi293F, respectively. These differences in pI were eliminated by enzymatic treatment with sialidase, indicating a large difference in the incorporation of sialic acid between hosts. This dramatic difference in the number of sialylated glycans between hosts was confirmed by analysis of PNGase F-released glycans using MALDI-ToF MS. These differences in glycosylation, however, did not greatly translate into differences in antibody recognition. Biosensor assays showed that gp145 produced in CHO-K1 had similar affinity toward the broadly neutralizing antibodies, 2G12 and PG16, as the gp145 produced in Expi293F. Additionally, both immunogens showed the same reactivity against plasma of HIV-infected patients. Taken together, these results support the notion that there are sizeable differences in the glycosylation of Env depending on the expression host. How these differences translate to vaccine efficacy remains unknown.

Antigenic competition in CD4+ T cell responses in a randomized, multicenter, double-blind clinical HIV vaccine trial.

T cell responses have been implicated in reduced risk of HIV acquisition in uninfected persons and control of viral replication in HIV-infected individuals. HIV Gag-specific T cells have been predominantly associated with post-infection control, whereas Env antigens are the target for protective antibodies; therefore, inclusion of both antigens is common in HIV vaccine design. However, inclusion of multiple antigens may provoke antigenic competition, reducing the potential effectiveness of the vaccine. HVTN 084 was a randomized, multicenter, double-blind phase 1 trial to investigate whether adding Env to a Gag/Pol vaccine decreases the magnitude or breadth of Gag/Pol-specific T cell responses. Fifty volunteers each received one intramuscular injection of 1 × 1010 particle units (PU) of rAd5 Gag/Pol and EnvA/B/C (3:1:1:1 mixture) or 5 × 109 PU of rAd5 Gag/Pol. CD4+ T cell responses to Gag/Pol measured 4 weeks after vaccination by cytokine expression were significantly higher in the group vaccinated without Env, whereas CD8+ T cell responses did not differ significantly between the two groups. Mapping of individual epitopes revealed greater breadth of the Gag/Pol-specific T cell response in the absence of Env compared to Env coimmunization. Addition of an Env component to a Gag/Pol vaccine led to reduced Gag/Pol CD4+ T cell response rate and magnitude as well as reduced epitope breadth, confirming the presence of antigenic competition. Therefore, T cell-based vaccine strategies should aim at choosing a minimalist set of antigens to reduce interference of individual vaccine components with the induction of the maximally achievable immune response.

HIV controllers suppress viral replication and evolution and prevent disease progression following intersubtype HIV-1 superinfection.

OBJECTIVE: The aim of this study was to investigate the impact of intersubtype HIV-1 superinfection on viremia, reservoir reseeding, viral evolution and disease progression in HIV controllers (HIC). DESIGN: A longitudinal analysis of two Brazilian HIC individuals (EEC09 and VC32) previously identified as dually infected with subtypes B and F1 viruses. METHODS: Changes in plasma viremia, total HIV-1 DNA levels, CD4+ T-cell counts and HIV-1 quasispecies composition were measured over time. HIV-1 env diversity in peripheral blood mononuclear cell (PBMC) and plasma samples was accessed by single genome amplification and next-generation sequencing approaches, respectively. Viral evolution was evaluated by estimating nucleotide diversity and divergence. RESULTS: Individual EEC09 was probably initially infected with a CCR5-tropic subtype B strain and sequentially superinfected with a CXCR4-tropic subtype B strain and with a subtype F1 variant. Individual VC32 was infected with a subtype B strain and superinfected with a subtype F1 variant. The intersubtype superinfection events lead to a moderate increase in viremia and extensive turnover of viral population in plasma but exhibited divergent impact on the size and composition of cell-associated HIV DNA population. Both individuals maintained virologic control (<2000 copies/ml) and presented no evidence of viral evolution or immunologic progression for at least 2 years after the intersubtype superinfection event. CONCLUSION: These data revealed that some HIC are able to repeatedly limit replication and evolution of superinfecting viral strains of a different subtype with no signs of disease progression.

Selective cytotoxicity of a novel immunotoxin based on pulchellin A chain for cells expressing HIV envelope.

Immunotoxins (ITs), which consist of antibodies conjugated to toxins, have been proposed as a treatment for cancer and chronic infections. To develop and improve the ITs, different toxins such as ricin, have been used, aiming for higher efficacy against target cells. The toxin pulchellin, isolated from the Abrus pulchellus plant, has similar structure and function as ricin. Here we have compared two plant toxins, recombinant A chains from ricin (RAC) and pulchellin (PAC) toxins, for their ability to kill HIV Env-expressing cells. In this study, RAC and PAC were produced in E. coli, and chromatographically purified, then chemically conjugated to two different anti-HIV monoclonal antibodies (MAbs), anti-gp120 MAb 924 or anti-gp41 MAb 7B2. These conjugates were characterized biochemically and immunologically. Cell internalization was studied by flow cytometry and confocal microscopy. Results showed that PAC can function within an effective IT. The ITs demonstrated specific binding against native antigens on persistently HIV-infected cells and recombinant antigens on Env-transfected cells. PAC cytotoxicity appears somewhat less than RAC, the standard for comparison. This is the first report that PAC may have utility for the design and construction of therapeutic ITs, highlighting the potential role for specific cell targeting.

Monocyte-lymphocyte fusion induced by the HIV-1 envelope generates functional heterokaryons with an activated monocyte-like phenotype.

Enveloped viruses induce cell-cell fusion when infected cells expressing viral envelope proteins interact with target cells, or through the contact of cell-free viral particles with adjoining target cells. CD4+ T lymphocytes and cells from the monocyte-macrophage lineage express receptors for HIV envelope protein. We have previously reported that lymphoid Jurkat T cells expressing the HIV-1 envelope protein (Env) can fuse with THP-1 monocytic cells, forming heterokaryons with a predominantly myeloid phenotype. This study shows that the expression of monocytic markers in heterokaryons is stable, whereas the expression of lymphoid markers is mostly lost. Like THP-1 cells, heterokaryons exhibited FcγR-dependent phagocytic activity and showed an enhanced expression of the activation marker ICAM-1 upon stimulation with PMA. In addition, heterokaryons showed morphological changes compatible with maturation, and high expression of the differentiation marker CD11b in the absence of differentiation-inducing agents. No morphological change nor increase in CD11b expression were observed when an HIV-fusion inhibitor blocked fusion, or when THP-1 cells were cocultured with Jurkat cells expressing a non-fusogenic Env protein, showing that differentiation was not induced merely by cell-cell interaction but required cell-cell fusion. Inhibition of TLR2/TLR4 signaling by a TIRAP inhibitor greatly reduced the expression of CD11b in heterokaryons. Thus, lymphocyte-monocyte heterokaryons induced by HIV-1 Env are stable and functional, and fusion prompts a phenotype characteristic of activated monocytes via intracellular TLR2/TLR4 signaling.

SERINC as a Restriction Factor to Inhibit Viral Infectivity and the Interaction with HIV.

The serine incorporator 5 (SERINC5) is a recently discovered restriction factor that inhibits viral infectivity by preventing fusion. Retroviruses have developed strategies to counteract the action of SERINC5, such as the expression of proteins like negative regulatory factor (Nef), S2, and glycosylated Gag (glycoGag). These accessory proteins downregulate SERINC5 from the plasma membrane for subsequent degradation in the lysosomes. The observed variability in the action of SERINC5 suggests the participation of other elements like the envelope glycoprotein (Env) that modulates susceptibility of the virus towards SERINC5. The exact mechanism by which SERINC5 inhibits viral fusion has not yet been determined, although it has been proposed that it increases the sensitivity of the Env by exposing regions which are recognized by neutralizing antibodies. More studies are needed to understand the role of SERINC5 and to assess its utility as a therapeutic strategy.

Cross-Neutralizing Antibodies in HIV-1 Individuals Infected by Subtypes B, F1, C or the B/Bbr Variant in Relation to the Genetics and Biochemical Characteristics of the env Gene.

Various HIV-1 env genetic and biochemical features impact the elicitation of cross-reactive neutralizing antibodies in natural infections. Thus, we aimed to investigate cross-neutralizing antibodies in individuals infected with HIV-1 env subtypes B, F1, C or the B/Bbr variant as well as env characteristics. Therefore, plasma samples from Brazilian chronically HIV-1 infected individuals were submitted to the TZM-bl neutralization assay. We also analyzed putative N-glycosylation sites (PNGLs) and the size of gp120 variable domains in the context of HIV-1 subtypes prevalent in Brazil. We observed a greater breadth and potency of the anti-Env neutralizing response in individuals infected with the F1 or B HIV-1 subtypes compared with the C subtype and the variant B/Bbr. We observed greater V1 B/Bbr and smaller V4 F1 than those of other subtypes (p<0.005), however neither was there a correlation verified between the variable region length and neutralization potency, nor between PNLG and HIV-1 subtypes. The enrichment of W at top of V3 loop in weak neutralizing response viruses and the P in viruses with higher neutralization susceptibility was statistically significant (p = 0.013). Some other signatures sites were associated to HIV-1 subtype-specific F1 and B/Bbr samples might influence in the distinct neutralizing response. These results indicate that a single amino acid substitution may lead to a distinct conformational exposure or load in the association domain of the trimer of gp120 and interfere with the induction power of the neutralizing response, which affects the sensitivity of the neutralizing antibody and has significant implications for vaccine design.

Sequencing of Gag/Env association with HIV genotyping resolution and HIV-related epidemiologic studies of HIV in China.

HIV genotyping has led to conflicting results between laboratories. Therefore, identifying the most accurate gene combinations to sequence remains a priority. Datasets of Chinese HIV subtypes based on several markers and deposited in PubMed, Metstr, CNKI, and VIP databases between 2000 and 2015 were studied. In total, 9177 cases of amplification-positive samples from 26 provinces of China were collected and used to classify HIV subtypes based on eight individual genes or a combination thereof. CRF01_AE, CRF07_BC, CRF08_BC and B were the prevalent HIV subtypes in China, accounting for 84.07% of all genotypes. Gag/Env sequencing classified a greater number of HIV subtypes compared to other genes or combination of gene fragments. The geographical distribution of Gag and Gag/Env genotypes was similar to that observed with all genetic markers. Further principal component analysis showed a significantly different geographical distribution pattern of HIV in China for HIV genotypes detected with Gag/Env, which was in line with the distribution of all HIV genotypes in China. Gag/Env sequences had the highest diversity of the eight markers studied, followed by Gag and Gag/Pol/Env; Pol/Env polymorphisms were the least divergent. Gag/Env can serve as a high-resolution marker for HIV genotyping.

Comprehensive Characterization of HIV-1 Molecular Epidemiology and Demographic History in the Brazilian Region Most Heavily Affected by AIDS.

UNLABELLED: The high incidence of AIDS cases and the dominance of HIV-1 subtype C infections are two features that distinguish the HIV-1 epidemic in the two southernmost Brazilian states (Rio Grande do Sul [RS] and Santa Catarina [SC]) from the epidemic in other parts of the country. Nevertheless, previous studies on HIV molecular epidemiology were conducted mainly in capital cities, and a more comprehensive understanding of factors driving this unique epidemic in Brazil is necessary. Blood samples were collected from individuals in 13 municipalities in the Brazilian southern region. HIV-1 env and pol genes were submitted to phylogenetic analyses for assignment of subtype, and viral population phylodynamics were reconstructed by applying Skygrid and logistic coalescent models in a Bayesian analysis. A high prevalence of subtype C was observed in all sampled locations; however, an increased frequency of recombinant strains was found in RS, with evidence for new circulating forms (CRFs). In the SC state, subtype B and C epidemics were associated with distinct exposure groups. Although logistic models estimated similar growth rates for HIV-1 subtype C (HIV-1C) and HIV-1B, a Skygrid plot reveals that the former epidemic has been expanding for a longer time. Our results highlight a consistent expansion of HIV-1C in south Brazil, and we also discuss how heterosexual and men who have sex with men (MSM) transmission chains might have impacted the current prevalence of HIV-1 subtypes in this region. IMPORTANCE: The AIDS epidemic in south Brazil is expanding rapidly, but the circumstances driving this condition are not well known. A high prevalence of HIV-1 subtype C was reported in the capital cities of this region, in contrast to the subtype B dominance in the rest of the country. This study sought to comparatively investigate the HIV-1 subtype B and C epidemics by sampling individuals from several cities in the two states with the highest AIDS incidences in Brazil. Our analyses showed distinct epidemic growth curves for the two epidemics, and we also found evidence suggesting that separate transmission chains may be impacting the viral phylodynamics and the emergence of new recombinant forms.

HIV Envelope Trimer Specific Immune Response Is Influenced by Different Adjuvant Formulations and Heterologous Prime-Boost.

The development of a preventive vaccine against human immunodeficiency virus (HIV-1) infection is the most efficient method to control the epidemic. The ultimate goal is to develop a vaccine able to induce specific neutralizing, non-neutralizing antibodies and cellular mediated immunity (CMI). Humoral and CMI responses can be directed to glycoproteins that are normally presented as a trimeric spike on the virus surface (gp140). Despite safer, subunit vaccines are normally less immunogenic/effective and need to be delivered together with an adjuvant. The choice of a suitable adjuvant can induce effective humoral and CMI that utterly lead to full protection against disease. In this report, we established a hierarchy of adjuvant potency on humoral and CMI when admixed with the recombinant HIV gp140 trimer. We show that vaccination with gp140 in the presence of different adjuvants can induce high-affinity antibodies, follicular helper T cells and germinal center B cells. The data show that poly (I:C) is the most potent adjuvant to induce specific CMI responses evidenced by IFN-γ production and CD4+/CD8+ T cell proliferation. Furthermore, we demonstrate that combining some adjuvants like MPL plus Alum and MPL plus MDP exert additive effects that impact on the magnitude and quality of humoral responses while mixing MDP with poly (I:C) or with R848 had no impact on total IgG titers but highly impact IgG subclass. In addition, heterologous DNA prime- protein boost yielded higher IgG titers when compare to DNA alone and improved the quality of humoral response when compare to protein immunization as evidenced by IgG1/IgG2a ratio. The results presented in this paper highlight the importance of selecting the correct adjuvant-antigen combination to potentiate desired cells for optimal stimulation.

Worldwide Genetic Features of HIV-1 Env α4ß7 Binding Motif: The Local Dissemination Impact of the LDI Tripeptide.

BACKGROUND: HIV-1 gp120 binds to integrin α4ß7, a homing receptor of lymphocytes to gut-associated lymphoid tissues. This interaction is mediated by the LDI/V tripeptide encoded in the V2-loop. This tripeptide mimics similar motifs in mucosal addressin cellular adhesion molecule (MAdCAM) and vascular CAM (VCAM), the natural ligands of α4ß7. In this study, we explored the association of V2-loop LDI/V mimotopes with transmission routes and patterns of disease progression in HIV-infected adult and pediatric patients. HIV-1 env sequences available in the Los Alamos HIV Sequence database were included in the analyses. METHODS: HIV-1 V2-loop sequences generated from infected adults and infants from South and Southeast Brazil, and also retrieved from the Los Alamos database, were assessed for α4ß7 binding tripeptide composition. Chi-Square/Fisher Exact test and Mann Whitney U test were used for tripeptide comparisons. Shannon entropy was assessed for conservancy of the α4ß7 tripeptide mimotope. RESULTS: We observed no association between the tripeptide composition or conservation and virus transmission route or disease progression. However, LDI was linked to successful epidemic dissemination of HIV-1 subtype C in South America, and further to other expanding non-B subtypes in Europe and Asia. In Africa, subtypes showing increased LDV prevalence evidenced an ongoing process of selection toward LDI expansion, an observation also extended to subtype B in the Americas and Western Europe. CONCLUSIONS: The V2-loop LDI mimotope was conserved in HIV-1C from South America and other expanding subtypes across the globe, which suggests that LDI may promote successful dissemination of HIV at local geographic levels by means of increased transmission fitness.

Assessing the HIV-1 Epidemic in Brazilian Drug Users: A Molecular Epidemiology Approach.

Person who inject illicit substances have an important role in HIV-1 blood and sexual transmission and together with person who uses heavy non-injecting drugs may have less than optimal adherence to anti-retroviral treatment and eventually could transmit resistant HIV variants. Unfortunately, molecular biology data on such key population remain fragmentary in most low and middle-income countries. The aim of the present study was to assess HIV infection rates, evaluate HIV-1 genetic diversity, drug resistance, and to identify HIV transmission clusters in heavy drug users (DUs). For this purpose, DUs were recruited in the context of a Respondent-Driven Sampling (RDS) study in different Brazilian cities during 2009. Overall, 2,812 individuals were tested for HIV, and 168 (6%) of them were positive, of which 19 (11.3%) were classified as recent seroconverters, corresponding to an estimated incidence rate of 1.58%/year (95% CI 0.92-2.43%). Neighbor joining phylogenetic trees from env and pol regions and bootscan analyses were employed to subtype the virus from132 HIV-1-infected individuals. HIV-1 subtype B was prevalent in most of the cities under analysis, followed by BF recombinants (9%-35%). HIV-1 subtype C was the most prevalent in Curitiba (46%) and Itajaí (86%) and was also detected in Brasília (9%) and Campo Grande (20%). Pure HIV-1F infections were detected in Rio de Janeiro (9%), Recife (6%), Salvador (6%) and Brasília (9%). Clusters of HIV transmission were assessed by Maximum likelihood analyses and were cross-compared with the RDS network structure. Drug resistance mutations were verified in 12.2% of DUs. Our findings reinforce the importance of the permanent HIV-1 surveillance in distinct Brazilian cities due to viral resistance and increasing subtype heterogeneity all over Brazil, with relevant implications in terms of treatment monitoring, prophylaxis and vaccine development.

Molecular Characterization of the Human Immunodeficiency Virus Type 1 in Women and Their Vertically Infected Children.

Approximately 35 million people worldwide are infected with human immunodeficiency virus (HIV) around 3.2 million of whom are children under 15 years. Mother-to-child-transmission (MTCT) of HIV-1 accounts for 90% of all infections in children. Despite great advances in the prevention of MTCT in Brazil, children are still becoming infected. Samples from 19 HIV-1-infected families were collected. DNA was extracted and fragments from gag, pol, and env were amplified and sequenced directly. Phylogenetic reconstruction was performed. Drug resistance analyses were performed in pol and env sequences. We found 82.1% of subtype B and 17.9% of BF recombinants. A prevalence of 43.9% drug resistance-associated mutations in pol sequences was identified. Of the drug-naive children 33.3% presented at least one mutation related to protease inhibitor/nucleoside reverse transcriptase inhibitor/nonnucleoside reverse transcriptase inhibitor (PI/NRTI/NNRTI) resistance. The prevalence of transmitted drug resistance mutations was 4.9%. On env we found a low prevalence of HR1 (4.9%) and HR2 (14.6%) mutations.

CRF19_cpx is an Evolutionary fit HIV-1 Variant Strongly Associated With Rapid Progression to AIDS in Cuba.

BACKGROUND: Clinicians reported an increasing trend of rapid progression (RP) (AIDS within 3 years of infection) in Cuba. METHODS: Recently infected patients were prospectively sampled, 52 RP at AIDS diagnosis (AIDS-RP) and 21 without AIDS in the same time frame (non-AIDS). 22 patients were sampled at AIDS diagnosis (chronic-AIDS) retrospectively assessed as > 3 years infected. Clinical, demographic, virological, epidemiological and immunological data were collected. Pol and env sequences were used for subtyping, transmission cluster analysis, and prediction of resistance, co-receptor use and evolutionary fitness. Host, immunological and viral predictors of RP were explored through data mining. FINDINGS: Subtyping revealed 26 subtype B strains, 6 C, 6 CRF18_cpx, 9 CRF19_cpx, 29 BG-recombinants and other subtypes/URFs. All patients infected with CRF19 belonged to the AIDS-RP group. Data mining identified CRF19, oral candidiasis and RANTES levels as the strongest predictors of AIDS-RP. CRF19 was more frequently predicted to use the CXCR4 co-receptor, had higher fitness scores in the protease region, and patients had higher viral load at diagnosis. INTERPRETATION: CRF19 is a recombinant of subtype D (C-part of Gag, PR, RT and nef), subtype A (N-part of Gag, Integrase, Env) and subtype G (Vif, Vpr, Vpu and C-part of Env). Since subtypes D and A have been associated with respectively faster and slower disease progression, our findings might indicate a fit PR driving high viral load, which in combination with co-infections may boost RANTES levels and thus CXCR4 use, potentially explaining the fast progression. We propose that CRF19 is evolutionary very fit and causing rapid progression to AIDS in many newly infected patients in Cuba.

[Use of a synthetic peptide of HIV-2 glycoprotein 36 in antigen mixtures for the immunodiagnosis of HIV types 1 and 2]. / Uso de un péptido sintético de la glucoproteína 36 del VIH 2 en mezclas de antígenos para el inmunodiagnóstico de la infección por VIH tipo 1 y 2.

INTRODUCTION: The HIV-2 glycoprotein 36 (gp36) is often used in ELISA. An evaluation of the diagnostic indexes of antigen mixtures with a synthetic peptide of HIV2 gp36 (5) is performed in this study. METHODS: Five mixtures of gp36 (5) and the recombinant proteins of HIV1/2 were prepared. A total of 1306 samples were evaluated with UMELISA HIV1+2 RECOMBINANT used as reference. RESULTS: The variant (V-1) showed very good agreement as regards the reference method. CONCLUSION: The V-1 variant was shown to be highly effective in the immunodiagnosis of HIV 1/2.

An HIV-1 transmission case possibly associated with manicure care.

A recently diagnosed 22-year-old female with no history of transmission risk factors prompted a thorough investigation of possible alternative risk factors. As the patient had evidence of advanced disease and laboratory data compatible with long-standing infection, past events were reviewed. About 10 years ago the patient shared manicure utensils with an older cousin, later known to be HIV infected; this prompted the phylogenetic analysis of the HIV sequences of both patients. Phylogenetic analyses of partial HIV-1 polymerase and envelope sequences from both patients revealed highly related sequences, with an estimated common ancestor date (about 11 years ago) that coincided with the putative sharing of manicure instruments, during a time in which the cousin was not virally suppressed. Taken together, the information about the infection of this patient suggests the use of shared manicure instruments as an alternative route of fomite HIV-1 transmission.

Phylogenetic analysis of human immunodeficiency virus type 2 isolated from Cuban individuals.

The presence of infection by human immunodeficiency virus type 2 (HIV-2) in Cuba has been previously documented. However, genetic information on the strains that circulate in the Cuban people is still unknown. The present work constitutes the first study concerning the phylogenetic relationship of HIV-2 Cuban isolates conducted on 13 Cuban patients who were diagnosed with HIV-2. The env sequences were analyzed for the construction of a phylogenetic tree with reference sequences of HIV-2. Phylogenetic analysis of the env gene showed that all the Cuban sequences clustered in group A of HIV-2. The analysis indicated several independent introductions of HIV-2 into Cuba. The results of the study will reinforce the program on the epidemiological surveillance of the infection in Cuba and make possible further molecular evolutionary studies.

Genotypic tropism prediction from paired cell and plasma using single and replicate sequences.

HIV-1 tropism determination is necessary prior to CCR5 antagonist use as antiretroviral therapy. Genotypic prediction of coreceptor use is a practical alternative to phenotypic tests. Cell DNA and plasma RNA-based prediction has shown discordance in many studies. We evaluate paired cell and plasma either as single or replicate V3 sequences to assess prediction comparability. The HIV-1 partial env region was sequenced and tropism was predicted using geno2pheno and position-specific scoring matrices (PSSM). Nucleotide ambiguities at V3 were quantified and genetic distance (Protdist) was determined using BioEdit. Wilcoxon signed-rank test, t tests, and Spearman correlation were performed with Prism GraphPad5.0. Results are expressed as medians, with a level of significance of p<0.05, two tailed. Single (n=28) or replicate (n=26) paired cell/plasma sequences were obtained from 54 patients. Although the clonalfalse-positive rate (FPR) value from both compartments strongly correlated (r=0.86 p<0.0001), discordance in tropism prediction was observed in both singles and replicates using geno2pheno or PSSM. Applying clonalFPR(10%) 46% (25/54) were X4 tropic, with a plasma/cell discordance of 11% in singles and 23% in replicates. Genetic distance (p<0.0001) and clonalFPR value dispersion (p=0.003) were significantly higher among replicate sequences from cells. Discordance of viral tropism prediction is not uncommon and the use of replicates does not decrease its occurrence, but improves X4 sensitivity. Sequences from provirus had greater genetic distance and dispersion of clonalFPR values. This may suggest that DNA replicate assays may better represent the diversity of HIV-1 variants, but the clinical significance of these findings needs further evaluation.

Replacement of the V3 domain in the surface subunit of the feline immunodeficiency virus envelope glycoprotein with the equivalent region of a T cell-tropic human immunodeficiency virus type 1 results in a chimeric surface protein that efficiently binds to CXCR4.

Feline immunodeficiency virus (FIV) and the T cell-tropic strains of human immunodeficiency virus type 1 (HIV-1) share the use of the chemokine receptor CXCR4 for cell entry. To study this process further we developed a cell surface binding assay based on the expression of a soluble version of the FIV SU C-terminally tagged with the influenza virus hemagglutinin epitope (HA). The specificity of the assay was demonstrated by the following evidence: (1) the SU-HA protein bound to HeLa cells that express CXCR4 but not to MDCK cells that lack this chemokine receptor; and (2) binding of the SU-HA to HeLa cells was blocked by incubation with the CXCR4 antagonist AMD3100 as well as with the anti-CXCR4 monoclonal antibody (MAb) 12G5. Deletion of the V3 region from the FIV SU glycoprotein abolished its ability to bind CXCR4-expressing cells. Remarkably, substitution of the V3 domain of the FIV SU by the equivalent region of the HIV-1 NL4-3 isolate resulted in efficient cell surface binding of the chimeric SU protein to CXCR4. Moreover, transfection of MDCK cells with a plasmid encoding human CXCR4 allowed the association of the chimeric SU-HA glycoprotein to the transfected cells. Interestingly, while cell binding of the chimeric FIV-HIV SU was inhibited by an anti-HIV-1 V3 MAb, its association with CXCR4 was found to be resistant to AMD3100. Of note, the chimeric FIV-HIV Env glycoprotein was capable of promoting CXCR4-dependent cell-to-cell fusion.