Recombinant Human Immunodeficiency Virus Research Articles

Purification of recombinant vesicular stomatitis virus-based HIV vaccine candidate.

In this work, laboratory- and large-scale methods were tested for purification of a human immunodeficiency virus (HIV) vaccine candidate, based on recombinant vesicular stomatitis virus (rVSV). First step of the purification, the clarification of the rVSVs produced in serum-free cell culture medium, was tested by centrifugation and filtration using different filtration media and pore sizes (0.45 to 30µm). To reduce the supernatant volume and process time, the clarified sample was concentrated by ultrafiltration either using tangential flow filtration or centrifugal-based filtration units, depending on the process scale. The final purification step at laboratory-scale, was carried out by density gradient ultracentrifugation, the recovery of which was compared with chromatographic purification at large-scale. The virus preparations were analyzed using dynamic light scattering to verify the virus size and transmission electron microscopy for purity and virus morphology. Density gradient ultracentrifugation allowed the recovery of≥80% infectious particles and reduced the contaminant DNA and host cell proteins relatively to standard ultracentrifugation pelleting using a sucrose cushion. At large-scale, weak and strong anion-exchangers were tested and compared. The best columns allowed infectious virus recoveries as high as 77% and eliminated 92% of host cell proteins.

Identification of N-glycans with GalNAc-containing antennae from recombinant HIV trimers by ion mobility and negative ion fragmentation

Negative ion collision-induced dissociation (CID) of underivatized N-glycans has proved to be a simple, yet powerful method for their structural determination. Recently, we have identified a series of such structures with GalNAc rather than the more common galactose capping the antennae of hybrid and complex glycans. As part of a series of publications describing the negative ion fragmentation of different types of N-glycan, this paper describes their CID spectra and estimated nitrogen cross sections recorded by travelling wave ion mobility mass spectrometry (TWIMS). Most of the glycans were derived from the recombinant glycoproteins gp120 and gp41 from the human immunodeficiency virus (HIV), recombinantly derived from human embryonic kidney (HEK 293T) cells. Twenty-six GalNAc-capped hybrid and complex N-glycans were identified by a combination of TWIMS, negative ion CID, and exoglycosidase digestions. They were present as the neutral glycans and their sulfated and α2→3-linked sialylated analogues. Overall, negative ion fragmentation of glycans generates fingerprints that reveal their structural identity.

Pseudomonas aeruginosa accentuates inflammatory responses under HIV infection through TREM‐1 activation

ObjectiveHuman immunodeficiency virus (HIV) threatens human health worldwide. Anti‐retroviral therapy (ART) extends survival but does not eliminate HIV from its cellular reservoirs. Patients accepting ART show altered immunity and immune activation, and continue to present increased risk of bacteria associated pneumonia, which is a major cause of morbidity and mortality. As an important opportunistic human pathogen, Pseudomonas aeruginosa causes a wide variety of severe nosocomial infections. Triggering receptor expressed on myeloid cells‐1 (TREM‐1) is an amplifier of inflammation, and has been recognized as a critical immunomodulator in inflammatory diseases of both infectious and non‐infectious etiologies. However, molecular mechanisms underlying the functional role of TREM‐1 in lung infections of HIV patients remains to be defined.MethodsWild type C57 BL/6J and HIV transgenic mice were treated with P. aeruginosa strain PAO1 through intranasal administration, mouse lung injury status was observed through Hematoxylin and Eosin (H&E) staining. Total RNA of lung tissues was isolated, real‐time RT‐PCR and western immunoblots were performed to quantify gene and protein expression. THP‐1 derived macrophages and human monocyte‐derived macrophages were treated with recombinant HIV viral proteins Tat and gp120, expression of miR‐155 and SOCS1 were quantified, and oxidative phosphorylation (OXPHOS) was measured through mitostress assay. Co‐culture model was used to analyze intracellular communications between macrophage and lung epithelial cells, and exosomes secreted by macrophages were isolated and cocultured with recipient cells, then expression of key tight junction proteins involved in epithelial barrier function were analyzed.ResultsCompared to wild type control, PAO1 accentuates acute neutrophilic inflammation in HIV transgenic mice with increase in neutrophil influx, expression levels of TREM‐1 and proinflammatory cytokines, particularly IL‐1β, TNF‐α and IL‐6. TREM‐1 silencing in macrophages exposed to HIV‐related proteins Tat or gp120, resulted in significant attenuation of miR‐155 expression with increased SOCS1 expression, and reduced mitochondrial reactive oxygen species (mtROS) and elevated oxygen consumption rate (OCR). Furthermore, TREM‐1 silencing in Tat‐treated macrophages reversed the disruption of tight junction barrier integrity in co‐cultured lung epithelial cells through modulation of macrophage‐derived exosome shuttling.ConclusionTREM‐1 escalates neutrophilic lung inflammation in HIV transgenic mice treated with PAO1, results in mitochondrial dysfunction of macrophages and disrupts tight junction barrier integrity of epithelia cells in lung microenvironment. These data suggest that TREM‐1 inhibition might be an important immunomodulatory therapeutic strategy for HIV‐infected patients with lung infections.Support or Funding InformationThis work is supported by VA Merit Review 2I01BX001786‐06A1 (Ruxana T Sadikot) and R01 HL125042(David M Guidot).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of the membrane-bound form of the chimeric, B/C recombinant HIV-1 Env, LT5.J4b12C.

Human immunodeficiency virus 1 (HIV-1) diversity is a significant challenge in developing a vaccine against the virus. B/C recombinants have been found in India and other places but are the predominant clade prevalent in China. HIV-1 envelopes (Envs) are the target of broadly neutralizing antibodies (bNAbs) which develop spontaneously in some HIV-1 infected patients. It has been previously reported with efficiently cleaved clade A, B and C Envs that preferential binding of Envs to bNAbs as opposed to non-NAbs, a desirable property for immunogens, is correlated with efficient cleavage of the Env precursor polypeptide into constituent subunits. These Envs are suitable for designing immunogens as soluble proteins, virus-like particles or for delivery by viral vectors/plasmid DNA. However, a B/C recombinant Env with similar properties has not been reported. Here we show that the chimeric, recombinant B/C clade Env LT5.J4b12C is efficiently cleaved on the plasma membrane and selectively binds to bNAbs.

Expression, purification, characterization and immunogenicity of human immunodeficiency virus-1 glycoprotein gp120 derived from insect cells

Objective To establish an efficient baculovirus-insect cell expression system for the production of human immunodeficiency virus-1 (HIV-1) envelope glycoprotein gp120 and to evaluate the physiochemical properties, antigenicity and immunogenicity of the recombinant protein. Methods The gene encoding HIV-1 NL4-3 gp120 was cloned into the downstream of pH promoter of the baculovirus transfer vector pAcgp67B to construct the recombinant transfer vector pAc-gp120. Expression of the protein of interest was induced in baculovirus-infected High Five™ insect cells. ELISA, analytical ultracentrifugation and size-exclusion chromatography were carried out to characterize physicochemical properties of the expressed gp120 protein. Immunogenicity of the recombinant gp120 protein was analyzed by HIV neutralization assay after immunizing BALB/c mice with it. Results The recombinant HIV-1 gp120 protein was successfully obtained from the established insect cell expression system with a purity of more than 90% and a mean yield of 13 mg/L in four batches. That recombinant HIV-1 gp120 protein was characterized by homogeneity in solution and possessed a good immunoreactivity to neutralizing antibodies and antisera against HIV. Immunogenicity analysis in BALB/c mice demonstrated that the recombinant gp120 protein could induce effective immune responses against HIV-1 NL4-3. Conclusion A simple and scalable approach to obtain homogeneous and immunogenic HIV-1 gp120 antigen is successfully established, which will promote further investigation of HIV vaccine candidates. Key words: Human immunodeficiency virus-1; Insect cell expression system; Glycoprotein; gp120; Immune response

Expression, purification, and renaturation of a recombinant peptide-based HIV vaccine in Escherichia coli.

To design an epitope-based vaccine for Human immunodeficiency virus (HIV), we previously predicted 20 potential HIV epitopes using bioinformatics approaches. The combination of these 20 epitopes has a theoretical coverage of 98.1% of the population for both the prevalent HIV genotypes and Chinese human leukocyte antigen DR types. To test the immunogenicity of this vaccine in vivo, a corresponding antigen needs to be prepared. To this end, we constructed a recombinant plasmid containing DNA encoding the epitopes and GPGPG spacers and a 6-His tag for verification of protein expression and ease of purification, and then transformed Escherichia coli cells with the plasmid. After IPTG induction, the recombinant protein was expressed in the form of mainly inclusion bodies. To stabilize the structure of denatured inclusion bodies for efficient purification and renaturation in vitro, we transferred the dissolved inclusion bodies from 7 mol/L guanidine hydrochloride to 8 mol/L urea. Under denaturing conditions, the vaccine protein was purified by a 3-step process including ion-exchange chromatography and affinity column, and then renatured by stepwise dialysis. Together, the above described procedures generated 43 mg of vaccine protein per litre of fermentation medium, and the final product reached approximately 95% purity. The purified protein was capable of eliciting antigen-specific T-cell responses in immunized mice.

Field-deployable, quantitative, rapid identification of active Ebola virus infection in unprocessed blood.

The West African Ebola virus outbreak underlined the importance of delivering mass diagnostic capability outside the clinical or primary care setting in effectively containing public health emergencies caused by infectious disease. Yet, to date, there is no solution for reliably deploying at the point of need the gold standard diagnostic method, real time quantitative reverse transcription polymerase chain reaction (RT-qPCR), in a laboratory infrastructure-free manner. In this proof of principle work, we demonstrate direct performance of RT-qPCR on fresh blood using far-red fluorophores to resolve fluorogenic signal inhibition and controlled, rapid freeze/thawing to achieve viral genome extraction in a single reaction chamber assay. The resulting process is entirely free of manual or automated sample pre-processing, requires no microfluidics or magnetic/mechanical sample handling and thus utilizes low cost consumables. This enables a fast, laboratory infrastructure-free, minimal risk and simple standard operating procedure suited to frontline, field use. Developing this novel approach on recombinant bacteriophage and recombinant human immunodeficiency virus (HIV; Lentivirus), we demonstrate clinical utility in symptomatic EBOV patient screening using live, infectious Filoviruses and surrogate patient samples. Moreover, we evidence assay co-linearity independent of viral particle structure that may enable viral load quantification through pre-calibration, with no loss of specificity across an 8 log-linear maximum dynamic range. The resulting quantitative rapid identification (QuRapID) molecular diagnostic platform, openly accessible for assay development, meets the requirements of resource-limited countries and provides a fast response solution for mass public health screening against emerging biosecurity threats.

Native Conformation and Canonical Disulfide Bond Formation Are Interlinked Properties of HIV-1 Env Glycoproteins.

We investigated whether there is any association between a native-like conformation and the presence of only the canonical (i.e., native) disulfide bonds in the gp120 subunits of a soluble recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein. We used a mass spectrometry (MS)-based method to map the disulfide bonds present in nonnative uncleaved gp140 proteins and native-like SOSIP.664 trimers based on the BG505 env gene. Our results show that uncleaved gp140 proteins were not homogeneous, in that substantial subpopulations (20 to 80%) contained aberrant disulfide bonds. In contrast, the gp120 subunits of the native-like SOSIP.664 trimer almost exclusively retained the canonical disulfide bond pattern. We also observed that the purification method could influence the proportion of an Env protein population that contained aberrant disulfide bonds. We infer that gp140 proteins may always contain a variable but substantial proportion of aberrant disulfide bonds but that the impact of this problem can be minimized via design and/or purification strategies that yield native-like trimers. The same factors may also be relevant to the production and purification of monomeric gp120 proteins that are free of aberrant disulfide bonds. It is widely thought that a successful HIV-1 vaccine will include a recombinant form of the Env protein, a trimer located on the virion surface. To increase yield and simplify purification, Env proteins are often made in truncated, soluble forms. A consequence, however, can be the loss of the native conformation concomitant with the virion-associated trimer. Moreover, some soluble recombinant Env proteins contain aberrant disulfide bonds that are not expected to be present in the native trimer. To assess whether these observations are linked, to determine the extent of disulfide bond scrambling, and to understand why scrambling occurs, we determined the disulfide bond profiles of two soluble Env proteins with different designs that are being assessed as vaccine candidates. We found that uncleaved gp140 forms heterogeneous mixtures in which aberrant disulfide bonds abound. In contrast, BG505 SOSIP.664 trimers are more homogeneous, native-like entities that contain predominantly the native disulfide bond profile.

Cleavage-independent HIV-1 Env trimers engineered as soluble native spike mimetics for vaccine design.

Viral glycoproteins mediate entry by pH-activated or receptor-engaged activation and exist in metastable pre-fusogenic states that may be stabilized by directed rational design. As recently reported, the conformationally fixed HIV-1 envelope glycoprotein (Env) trimers in the pre-fusion state (SOSIP) display molecular homogeneity and structural integrity at relatively high levels of resolution. However, the SOSIPs necessitate full Env precursor cleavage, which requires endogenous furin overexpression. Here, we developed an alternative strategy using flexible peptide covalent linkage of Env subdomains to produce soluble, homogeneous, and cleavage-independent Env mimics, called native flexibly linked (NFL) trimers, as vaccine candidates. This simplified design avoids the need for furin co-expression and, in one case, antibody affinity purification to accelerate trimer scale-up for preclinical and clinical applications. We have successfully translated the NFL design to multiple HIV-1 subtypes, establishing the potential to become a general method of producing native-like, well-ordered Env trimers for HIV-1 or other viruses.

GS-8374, a Prototype Phosphonate-Containing Inhibitor of HIV-1 Protease, Effectively Inhibits Protease Mutants with Amino Acid Insertions

Insertions in the protease (PR) region of human immunodeficiency virus (HIV) represent an interesting mechanism of antiviral resistance against HIV PR inhibitors (PIs). Here, we demonstrate the improved ability of a phosphonate-containing experimental HIV PI, GS-8374, relative to that of other PIs, to effectively inhibit patient-derived recombinant HIV strains bearing PR insertions and numerous other mutations. We correlate enzyme inhibition with the catalytic activities of corresponding recombinant PRs in vitro and provide a biochemical and structural analysis of the PR-inhibitor complex.

MALDI-MS/MS with Traveling Wave Ion Mobility for the Structural Analysis of N-Linked Glycans

The preference for singly charged ion formation by MALDI makes it a better choice than electrospray ionization for profiling mixtures of N-glycans. For structural analysis, fragmentation of negative ions often yields more informative spectra than fragmentation of positive ones but such ions are more difficult to produce from neutral glycans under MALDI conditions. This work investigates conditions for the formation of both positive and negative ions by MALDI from N-linked glycans released from glycoproteins and their subsequent MS/MS and ion mobility behaviour. 2,4,6-Trihydroxyacetophenone (THAP) doped with ammonium nitrate was found to give optimal ion yields in negative ion mode. Ammonium chloride or phosphate also yielded prominent adducts but anionic carbohydrates such as sulfated N-glycans tended to ionize preferentially. Carbohydrates adducted with all three adducts (phosphate, chloride, and nitrate) produced good negative ion CID spectra but those adducted with iodide and sulfate did not yield fragment ions although they gave stronger signals. Fragmentation paralleled that seen following electrospray ionization providing superior spectra than could be obtained by PSD on MALDI-TOF instruments or with ion traps. In addition, ion mobility drift times of the adducted glycans and the ability of this technique to separate isomers also mirrored those obtained following ESI sample introduction. Ion mobility also allowed profiles to be obtained from samples whose MALDI spectra showed no evidence of such ions allowing the technique to be used in conditions where sample amounts were limiting. The method was applied to N-glycans released from the recombinant human immunodeficiency virus glycoprotein, gp120.

Subtype analysis of gp41 gene of HIV-1 among men who have sex with men in Zhengzhou

This study aimed to investigate the subtype distribution of gp41 gene of human immunodeficiency virus-1 (HIV-1) among men who have sex with men (MSM) in Zhengzhou. Thirty blood samples were collected from men who have sex with men infected by HIV. The complete gp41 gene was amplified by RT-PCR and nested-PCR and sequenced. All sequences were edited by Bioedit and subtyped with HIV sequence library US Los Alamos National Laboratory and online genotyping software provided by American National Center of Biotechnology Information. Phylogenetic analysis of gp41 gene was performed using the MEGA 3.1 software, and the genic dispersion rates among subtype of gp41 gene were analyzed. A total of eighteen gene sequences of HIV-1 gp41 gene were obtained from thirty men who have sex with men infected by HIV, which belonged to subtype CRF15-01B (50% (9/18)), CRF01-AE (22% (4/18)), CRF07-B (22% (4/18)) and B (6% (1/18)), respectively. The intersubtype HIV-1 strains aggregate with according reference strains. The genetic distance inter-subtype of subtype CRF15-01B, CRF01-AE and CRF07-B were 0.050 ± 0.007, 0.052 ± 0.009 and 0.082 ± 0.012, respectively. The prevalent subtypes of HIV-1 among among MSM in Zhengzhou was complicated and recombinant HIV-1 strains were the most prevalent strains.

Epstein–Barr virus genome load is increased by therapeutic vaccination in HIV-l carriers, and further enhanced in patients with a history of symptomatic primary infection

ObjectiveEpstein–Barr virus (EBV) infection is an established risk factor for B-cell lymphomas in Human Immunodeficiency virus (HIV)-1 infected patients. A disturbed EBV-host relationship is seen in patient groups with a high risk for EBV-associated lymphomas. We have analysed this relationship by measuring EBV-DNA in the blood of HIV-1 carriers. MethodEBV-DNA load in B-cells was monitored by PCR in non- or insufficiently antiretroviral treated and rgp160-vaccinated HIV-patients. ResultsBoth asymptomatic HIV-infected and AIDS-patients showed a 25–40-fold increase in the number of B cell associated EBV-DNA copies compared to healthy controls. Patients included in a vaccine trial with recombinant HIV gp160 showed a 5-fold increase of EBV load compared to non-immunised patients and a 50-fold increase compared to healthy controls. There was no difference whether they received vaccine or “placebo”. Vaccinated patients with a history of symptomatic primary HIV-1 infection (PHI) had a 280-fold increase in median EBV load compared to healthy controls, thus suggesting a synergistic effect between the vaccination and PHI, which hypothetically could affect lymphoma risk. ConclusionsWe recommend analysis of EBV-load and long term follow up of lymphoma risk in all therapeutic HIV-1 vaccination trials.

Extended Follow-up Confirms Early Vaccine-Enhanced Risk of HIV Acquisition and Demonstrates Waning Effect Over Time Among Participants in a Randomized Trial of Recombinant Adenovirus HIV Vaccine (Step Study)

The Step Study tested whether an adenovirus serotype 5 (Ad5)-vectored human immunodeficiency virus (HIV) vaccine could prevent HIV acquisition and/or reduce viral load set-point after infection. At the first interim analysis, nonefficacy criteria were met. Vaccinations were halted; participants were unblinded. In post hoc analyses, more HIV infections occurred in vaccinees vs placebo recipients in men who had Ad5-neutralizing antibodies and/or were uncircumcised. Follow-up was extended to assess relative risk of HIV acquisition in vaccinees vs placebo recipients over time. We used Cox proportional hazard models for analyses of vaccine effect on HIV acquisition and vaccine effect modifiers, and nonparametric and semiparametric methods for analysis of constancy of relative risk over time. One hundred seventy-two of 1836 men were infected. The adjusted vaccinees vs placebo recipients hazard ratio (HR) for all follow-up time was 1.40 (95% confidence interval [CI], 1.03-1.92; P= .03). Vaccine effect differed by baseline Ad5 or circumcision status during first 18 months, but neither was significant for all follow-up time. The HR among uncircumcised and/or Ad5-seropositive men waned with time since vaccination. No significant vaccine-associated risk was seen among circumcised, Ad5-negative men (HR, 0.97; P=1.0) over all follow-up time. The vaccine-associated risk seen in interim analysis was confirmed but waned with time from vaccination.

Host–Epstein–Barr virus relationship affected by immunostimulation in HIV-infected patients representing distinct progressor profile groups

Human immunodeficiency virus (HIV) infection with pronounced immunosuppression disrupts Epstein-Barr virus (EBV)-host balance with increased lymphoma risk. We explored whether different host responses to HIV are reflected in the EBV-host balance. Eleven unvaccinated HIV-positive patients and 16 participants in a vaccine trial were included in the study. Blood samples were collected, B cells extracted, and EBV DNA load was determined using a semiquantitative polymerase chain reaction (PCR) method. Treatment-naïve patients with a history of symptomatic primary HIV infection showed non-significant, but higher EBV load compared to untreated long-term non-progressors. A significant difference in HIV RNA titres between these groups correlated weakly to EBV DNA load. Patients in the vaccine trial with recombinant HIV gp160 and/or adjuvant and with a history of symptomatic primary HIV infection, showed a 1-log increase in EBV load compared to patients with long-lasting HIV disease. Different host responses to HIV infection, especially in combination with vaccination, can be reflected in the EBV-host balance.

Fluorescent Reporter Signals, EGFP, and DsRed, Encoded in HIV-1 Facilitate the Detection of Productively Infected Cells and Cell-Associated Viral Replication Levels

Flow cytometric analysis is a reliable and convenient method for investigating molecules at the single cell level. Previously, recombinant human immunodeficiency virus type 1 (HIV-1) strains were constructed that express a fluorescent reporter, either enhanced green fluorescent protein, or DsRed, which allow the monitoring of HIV-1-infected cells by flow cytometry. The present study further investigated the potential of these recombinant viruses in terms of whether the HIV-1 fluorescent reporters would be helpful in evaluating viral replication based on fluorescence intensity. When primary CD4+ T cells were infected with recombinant viruses, the fluorescent reporter intensity measured by flow cytometry was associated with the level of CD4 downmodulation and Gag p24 expression in infected cells. Interestingly, some HIV-1-infected cells, in which CD4 was only moderately downmodulated, were reporter-positive but Gag p24-negative. Furthermore, when the activation status of primary CD4+ T cells was modulated by T cell receptor-mediated stimulation, we confirmed the preferential viral production upon strong stimulation and showed that the intensity of the fluorescent reporter within a proportion of HIV-1-infected cells was correlated with the viral replication level. These findings indicate that a fluorescent reporter encoded within HIV-1 is useful for the sensitive detection of productively infected cells at different stages of infection and for evaluating cell-associated viral replication at the single cell level.

Anti-retroviral Activity of GMP-Grade Stampidine against Genotypically and Phenotypically Nucleoside Reverse Transcriptase Inhibitor Resistant Recombinant Human Immunodeficiency Virus

The in vitro potency of GMP-grade stampidine (CAS 217178-62-6) was examined against 3 clinical HIV-1 isolates and 6 recombinant HIV-1 clones with multi-NRTI 'resistance (NRTI: nucleoside reverse transcriptase inhibitors). GMP-grade stampidine active drug substance (Lot #'s MPR-M0008.00-01 and MPR-M0008.01-01) as well as GMP-grade stampidine extracted from the clinical stampidine capsules (GMP-Grade Clinical Batch, Pharmaceutical Service Lot Number 159I0601) were highly potent and exhibited nanomolar IC50 values against clinical HIV-1 isolates as well as recombinant HIV-1 clones with multi-NRTI resistance containing common patterns of reverse transcriptase mutations responsible for NRTI resistance.

Predictors for Recombinant HIV Infection in a Thai Cohort

Recombinant human immunodeficiency virus (HIV) infection was associated with exchange of sex for money, ≥1 sex partner within the prior 6 months, and decline in CD4 cell count in this Thai cohort study. These findings suggest that recombinant HIV infection may have implications for HIV disease progression, safer sex practices, and vaccine development.

English

Serological methods are the main methods in diagnosis of human immunodeficiency virus (HIV)-1 and HIV-2 infection. The western Blot (WB) is considered as a gold standard test for the confirmation of enzyme-linked immunosorbent assay (ELISA) and/or other rapid screened reactive results samples in the diagnosis of HIV infection. However, problems of WB, particularly in regard to the significance of indeterminate results, still remain. To develop and evaluate a recombinant HIV-1/2 antigens-based line immunoassay as confirmatory assay for HIV-1 and HIV-2 infections is the objective of this study. HIV-1 envelope proteins (gp160, gp120, gp41), HIV-1 integrase protein (p31), HIV-1 gag proteins (p24, p17) and HIV-2 envelope protein (gp 36) were cloned and expressed inEscherichia coli. All recombinant HIV antigens were purified and immobilized on nitrocellulose membranes and applied in a line immunoassay, which supplement detected anti-HIV antibodies in a single strip. Specific antibodies to these antigens in human sera were revealed using an alkaline phosphatase labeled anti-human immunoglobulin G (IgG) as second antibody. Two anti-human IgG lines were used as a control for the addition of serum. Positive or negative results were determined by visual comparison of the antigen lines intensity with the two control lines. The recombinant HIV antigens-based line immunoassay identified seropositive individuals with a high degree of accuracy; none of the HIV-seropositive subjects yielded a negative result. Reactivity with these antigens, demonstrated 100% sensitivity and specificity in distinguishing seronegative from seropositive sera. This line immunoassay was much more sensitive and specific than WB. In particular, the line immunoassay allowed for a significant reduction in the number of indeterminate results and for more accurate distinction between HIV-1 and/or HIV-2 infections.   Key words: HIV antibodies, line immunoassay, recombinant antigens, confirmatory test.

On the Solution Conformation and Dynamics of the HIV-1 Viral Infectivity Factor

Human immunodeficiency virus-1 (HIV-1) has evolved a cunning mechanism to circumvent the antiviral activity of the APOBEC3 family of host cell enzymes. HIV-1 Vif [ viral (also called virion) infectivity factor], one of several HIV accessory proteins, targets APOBEC3 proteins for proteasomal degradation and downregulates their expression at the mRNA level. Despite the importance of Vif for HIV-1 infection, there is little conformational data on Vif alone or in complex with other cellular factors due to incompatibilities with many structural techniques and difficulties in producing suitable quantities of the protein for biophysical analysis. As an alternative, we have turned to hydrogen exchange mass spectrometry (HX MS), a conformational analysis method that is well suited for proteins that are difficult to study using X-ray crystallography and/or NMR. HX MS was used to probe the solution conformation of recombinant full-length HIV-1 Vif. Vif specifically interacted with the previously identified binding partner Hck and was able to cause kinase activation, suggesting that the Vif studied by HX MS retained a biochemically competent conformation relevant to Hck interaction. HX MS analysis of Vif alone revealed low deuteration levels in the N-terminal portion, indicating that this region contained structured or otherwise protected elements. In contrast, high deuteration levels in the C-terminal portion of Vif indicated that this region was likely unstructured in the absence of cellular interacting proteins. Several regions within Vif displayed conformational heterogeneity in solution, including the APOBEC3G/F binding site and the HCCH zinc finger. Taken together, these HX MS results provide new insights into the solution conformation of Vif.