Human Immunodeficiency Virus Gag Research Articles

Human Immunodeficiency Virus gag and protease: partners in resistance

Human Immunodeficiency Virus (HIV) maturation plays an essential role in the viral life cycle by enabling the generation of mature infectious virus particles through proteolytic processing of the viral Gag and GagPol precursor proteins. An impaired polyprotein processing results in the production of non-infectious virus particles. Consequently, particle maturation is an excellent drug target as exemplified by inhibitors specifically targeting the viral protease (protease inhibitors; PIs) and the experimental class of maturation inhibitors that target the precursor Gag and GagPol polyproteins. Considering the different target sites of the two drug classes, direct cross-resistance may seem unlikely. However, coevolution of protease and its substrate Gag during PI exposure has been observed both in vivo and in vitro. This review addresses in detail all mutations in Gag that are selected under PI pressure. We evaluate how polymorphisms and mutations in Gag affect PI therapy, an aspect of PI resistance that is currently not included in standard genotypic PI resistance testing. In addition, we consider the consequences of Gag mutations for the development and positioning of future maturation inhibitors.

A Small-molecule-controlled System for Efficient Pseudotyping of Prototype Foamy Virus Vectors

Foamy virus (FV) vector systems have recently demonstrated their power as efficient gene transfer tools for different target tissues. Unfortunately, FVs cannot be naturally pseudotyped by heterologous viral glycoproteins due to an unusual particle morphogenesis involving a FV Env-dependent particle release process. Therefore, current FV vector systems are constrained to the broad host cell range provided by the cognate viral glycoprotein. We evaluated different approaches for pseudotyping of FV vectors, in which the specific FV Gag-Env interaction, essential for particle egress, is substituted by a small-molecule controlled heterodimerization (HD) system. In one system developed, one HD-domain (HDD) is fused to a membrane-targeting domain (MTD), such as the human immunodeficiency virus (HIV) Gag matrix (MA) subunit, with a second fused to the FV capsid protein. Coexpression of both components with different heterologous viral glycoproteins allowed an efficient, dimerizer-dependent pseudotyping of FV capsids. With this system FV vesicular stomatitis virus glycoprotein (VSV-G) pseudotype titers greater than 1 × 10(6) IU/ml were obtained, at levels comparable to authentic FV vector particles. As a proof-of-principle we demonstrate that Pac2 cells, naturally resistant to FV vectors, become permissive to FV VSV-G pseudotypes. Similar to other retroviral vectors, this FV pseudotyping system now enables adaptation of cell-specific targeting approaches for FVs.

The Spread of HIV in Pakistan: Bridging of the Epidemic between Populations

In the last two decades, ‘concentrated epidemics’ of human immunodeficiency virus (HIV) have established in several high risk groups in Pakistan, including Injecting Drug Users (IDUs) and among men who have sex with men (MSM). To explore the transmission patterns of HIV infection in these major high-risk groups of Pakistan, 76 HIV samples were analyzed from MSM, their female spouses and children, along with 26 samples from a previously studied cohort of IDUs. Phylogenetic analysis of HIV gag gene sequences obtained from these samples indicated a substantial degree of intermixing between the IDU and MSM populations, suggesting a bridging of HIV infection from IDUs, via MSM, to the MSM spouses and children. HIV epidemic in Pakistan is now spreading to the female spouses and offspring of bisexual MSM. HIV control and awareness programs must be refocused to include IDUs, MSM, as well as bisexual MSM, and their spouses and children.

Identification of an inhibitory budding signal that blocks the release of HIV particles and exosome/microvesicle proteins

Animal cells bud exosomes and microvesicles (EMVs) from endosome and plasma membranes. The combination of higher-order oligomerization and plasma membrane binding is a positive budding signal that targets diverse proteins into EMVs and retrovirus particles. Here we describe an inhibitory budding signal (IBS) from the human immunodeficiency virus (HIV) Gag protein. This IBS was identified in the spacer peptide 2 (SP2) domain of Gag, is activated by C-terminal exposure of SP2, and mediates the severe budding defect of p6-deficient and PTAP-deficient strains of HIV. This IBS also impairs the budding of CD63 and several other viral and nonviral EMV proteins. The IBS does not prevent cargo delivery to the plasma membrane, a major site of EMV and virus budding. However, the IBS does inhibit an interaction between EMV cargo proteins and VPS4B, a component of the endosomal sorting complexes required for transport (ESCRT) machinery. Taken together, these results demonstrate that inhibitory signals can block protein and virus budding, raise the possibility that the ESCRT machinery plays a role in EMV biogenesis, and shed new light on the role of the p6 domain and PTAP motif in the biogenesis of HIV particles.

Human Immunodeficiency Virus (HIV) types Western blot (WB) band profiles as potential surrogate markers of HIV disease progression and predictors of vertical transmission in a cohort of infected but antiretroviral therapy naïve pregnant women in Harare, Zimbabwe

BackgroundExpensive CD4 count and viral load tests have failed the intended objective of enabling access to HIV therapy in poor resource settings. It is imperative to develop simple, affordable and non-subjective disease monitoring tools to complement clinical staging efforts of inexperienced health personnel currently manning most healthcare centres because of brain drain. Besides accurately predicting HIV infection, sequential appearance of specific bands of WB test offers a window of opportunity to develop a less subjective tool for monitoring disease progression.MethodsHIV type characterization was done in a cohort of infected pregnant women at 36 gestational weeks using WB test. Student-t test was used to determine maternal differences in mean full blood counts and viral load of mothers with and those without HIV gag antigen bands. Pearson Chi-square test was used to assess differences in lack of bands appearance with vertical transmission and lymphadenopathy.ResultsAmong the 64 HIV infected pregnant women, 98.4% had pure HIV-1 infection and one woman (1.7%) had dual HIV-1/HIV-2 infections. Absence of HIV pol antigen bands was associated with acute infection, p = 0.002. All women with chronic HIV-1 infection had antibody reactivity to both the HIV-1 envelope and polymerase antigens. However, antibody reactivity to gag antigens varied among the women, being 100%, 90%, 70% and 63% for p24, p17, p39 and p55, respectively. Lack of antibody reactivity to gag p39 antigen was associated with disease progression as confirmed by the presence of lymphadenopathy, anemia, higher viral load, p = 0.010, 0.025 and 0.016, respectively. Although not statistically significant, women with p39 band missing were 1.4 times more likely to transmit HIV-1 to their infants.ConclusionAbsence of antibody reactivity to pol and gag p39 antigens was associated with acute infection and disease progression, respectively. Apart from its use in HIV disease diagnosis, WB test could also be used in conjunction with simpler tests like full blood counts and patient clinical assessment as a relatively cheaper disease monitoring tool required prior to accessing antiretroviral therapy for poor resource settings. However, there is also need to factor in the role of host-parasite genetics and interactions in disease progression.

T-cell Epitopes Identified by BALB/c Mice Immunized with Vaccinia Expressing HIV-1 Gag lie within immunodominant Regions Recognized by HIV-infected Indian Patients.

Background:Human immunodeficiency virus (HIV) antigens from transmitted strains of HIV would prove crucial in vaccine designing for prevention of HIV infection. Immune response generated by Vaccinia construct expressing the HIV-1 gag gene from transmitted Indian HIV-1 subtype C strain (Vgag) in BALB/c mice is reported in the present study along with the identification of epitopes responsible for induction of the immune response.Aims:The aim of this study was to determine immune response generated by the constructs in a mouse model and to understand the epitope specificities of the response.Settings and Design:This was an observational study carried out in BALB/c mice.Materials and Methods:The immunogenecity of Vgag construct was evaluated in BALB/c mice after multiple immunizations. T-cell response was monitored by the interferon-γ ELISPOT assay using HIV-1 C Gag overlapping peptides and anti-P24 antibodies were estimated by ELISA.Statistical Analysis Used:Graphpad prism software was used for statistical analysis and for plotting graphs.Results:IFN-γ-secreting T cells and antibodies were detected against HIV Gag in mice after immunization. Although after repeated immunizations, antibody-mediated immune response increased or remained sustained, the magnitude of IFN-γ-secreting T cell was found to be decreased over time. The Gag peptides recognized by mice were mainly confined to the P24 region and had a considerable overlap with earlier reported immunodominant regions recognized by HIV-infected Indian patients.Conclusion:Vaccinia construct with a gag gene from transmitted HIV-1 virus was found to be immunogenic. The Gag regions identified by mice could have important implications in terms of future HIV vaccine designing.

Newcastle Disease Virus Expressing a Dendritic Cell-Targeted HIV Gag Protein Induces a Potent Gag-Specific Immune Response in Mice

Viral vaccine vectors have emerged as an attractive strategy for the development of a human immunodeficiency virus (HIV) vaccine. Recombinant Newcastle disease virus (rNDV) stands out as a vaccine vector since it has a proven safety profile in humans, it is a potent inducer of both alpha interferon (IFN-α) and IFN-β) production, and it is a potent inducer of dendritic cell (DC) maturation. Our group has previously generated an rNDV vector expressing a codon-optimized HIV Gag protein and demonstrated its ability to induce a Gag-specific CD8(+) T cell response in mice. In this report we demonstrate that the Gag-specific immune response can be further enhanced by the targeting of the rNDV-encoded HIV Gag antigen to DCs. Targeting of the HIV Gag antigen was achieved by the addition of a single-chain Fv (scFv) antibody specific for the DC-restricted antigen uptake receptor DEC205 such that the DEC205 scFv-Gag molecule was encoded for expression as a fusion protein. The vaccination of mice with rNDV coding for the DC-targeted Gag antigen induced an enhanced Gag-specific CD8(+) T cell response and enhanced numbers of CD4(+) T cells and CD8(+) T cells in the spleen relative to vaccination with rNDV coding for a nontargeted Gag antigen. Importantly, mice vaccinated with the DEC205-targeted vaccine were better protected from challenge with a recombinant vaccinia virus expressing the HIV Gag protein. Here we demonstrate that the targeting of the HIV Gag antigen to DCs via the DEC205 receptor enhances the ability of an rNDV vector to induce a potent antigen-specific immune response.

Intracellular localization of human immunodeficiency virus type 1 Gag and GagPol products and virus particle release: relationship with the Gag-to-GagPol ratio

Human immunodeficiency virus (HIV) Gag precursor protein is cleaved by viral protease (PR) within GagPol precursor protein to produce the mature matrix (MA), capsid, nucleocapsid, and p6 domains. This processing is termed maturation and required for HIV infectivity. In order to understand the intracellular sites and mechanisms of HIV maturation, HIV molecular clones in which Gag and GagPol were tagged with FLAG and hemagglutinin epitope sequences at the C-termini, respectively were made. When coexpressed, both Gag and GagPol were incorporated into virus particles. Temporal analysis by confocal microscopy showed that Gag and GagPol were relocated from the cytoplasm to the plasma membrane. Mature cleaved MA was observed only at sites on the plasma membrane where both Gag and GagPol had accumulated, indicating that Gag processing occurs during Gag/GagPol assembly at the plasma membrane, but not during membrane trafficking. Fluorescence resonance energy transfer imaging suggested that these were the primary sites of GagPol dimerization. In contrast, with overexpression of GagPol alone an absence of particle release was observed, and this was associated with diffuse distribution of mature cleaved MA throughout the cytoplasm. Alteration of the Gag-to-GagPol ratio similarly impaired virus particle release with aberrant distributions of mature MA in the cytoplasm. However, when PR was inactive, it seemed that the Gag-to-GagPol ratio was not critical for virus particle release but virus particles encasing unusually large numbers of GagPol molecules were produced, these particles displaying aberrant virion morphology. Taken together, it was concluded that the Gag-to-GagPol ratio has significant impacts on either intracellular distributions of mature cleaved MA or the morphology of virus particles produced.

Enhanced Induction of HIV-specific Cytotoxic T Lymphocytes by Dendritic Cell-targeted Delivery of SOCS-1 siRNA

Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in the activation of T cells. RNA interference (RNAi)-mediated silencing of negative immunoregulatory molecules expressed by DCs may provide a strategy to enhance the potency of DC-based vaccines and immunotherapy. Ablation of suppressor of cytokine signaling-1 (SOCS-1) in antigen-presenting cells has been shown to enhance cellular immune response in mice. Here, we used a previously reported DC-targeting approach to deliver small interfering RNA (siRNA) against SOCS-1 to human myeloid-derived DCs (MDDCs). SOCS1-silencing in MDDCs resulted in enhanced cytokine responses to lipopolysaccharide (LPS) and a strong mixed-lymphocyte reaction. Moreover, only DCs treated with SOCS-1 siRNA, and not controls, elicited strong primary in vitro responses to well-characterized HLA-A*0201-restricted Melan-A/MART-1 and human immunodeficiency virus (HIV) Gag epitopes in naive CD8(+) T cells from healthy donors. Finally, stimulation of CD8(+) T cells from HIV-seropositive subjects with SOCS1-silenced DCs resulted in an augmented polyfunctional cytotoxic T-lymphocyte (CTL) response, suggesting that SOCS-1 silencing can restore functionally compromised T cells in HIV infection. Collectively, these results demonstrate the feasibility of DC3-9dR-mediated manipulation of DC function to enhance DC immunogenicity for potential vaccine or immunotherapeutic applications.

Evaluation of the effect of pyrimethamine, an anti-malarial drug, on HIV-1 replication

Co-infection of human immunodeficiency virus (HIV) with malaria is one of the pandemic problems in Africa and parts of Asia. Here we investigated the impact of pyrimethamine (PYR) and two other clinical anti-malarial drugs (chloroquine [CQ] or artemisinin [ART]) on HIV-1 replication. Peripheral blood mononuclear cells (PBMCs) or MT-2 cells were infected with HIV NL4.3 strain and treated with different concentrations of the anti-malarial drugs. HIV-1 replication was measured using p24 ELISA. We show that 10 μM CQ and ART inhibited HIV-1 replication by 76% and 60% in PBMCs, respectively, but not in MT-2 cells. In contrast, 10 μM PYR enhanced HIV-1 replication in MT-2 cells by >10-fold. A series of molecular mechanism studies revealed that PYR increased intracellular HIV gag proteins without affecting the promoter or the reverse transcriptase activity. The effect of PYR was independent of HTLV-1 produced by MT-2 cells. Of interest, PYR treatment led to S-phase accumulation and increased AZT and d4T antiviral activity by ∼4-fold. Taken together, we show that PYR significantly enhances HIV-1 replication by affecting the cellular machinery. Our results could be relevant for the management of malaria and HIV particularly in regions where HIV-1 and malaria epidemics overlap.

Human immunodeficiency virus type I-specific CD8+T cell subset abnormalities in chronic infection persist through effective antiretroviral therapy

BackgroundEffective highly active antiretroviral therapy (HAART) reduces human immunodeficiency virus (HIV) replication, restores CD4+ T lymphocyte counts and greatly reduces the incidence of opportunistic infections. While this demonstrates improved generalized immune function, rapid rebound to pre-treatment viral replication levels following treatment interruption indicates little improvement in immune control of HIV replication. The extent to which HAART can normalize HIV-specific CD8+ T cell function over time in individuals with chronic infection remains an important unresolved issue. In this study, we evaluated the magnitude, general specificity and character of HIV specific CD8+ T cell responses at four time points across 2-9 years in 2 groups of chronically infected individuals separated on the basis of either effective antiretroviral suppression or ongoing replication of HIV.MethodsPeripheral blood mononuclear cells (PBMC) were stimulated with overlapping 15mer peptides spanning HIV Gag, Pol, Env and Nef proteins. Cells producing interferon-γ (IFN-γ) or interleukin-2 (IL-2) were enumerated by ELISPOT and phenotyped by flow cytometry.Results and ConclusionsThe magnitude of the HIV-specific CD8+ T cell response ranged from < .01 to approximately 1.0% of PBMC and was significantly greater in the group with detectable viral replication. Stronger responses reflected higher numbers of CD8+CD45RA- effector memory cells producing IFN-γ, but not IL-2. Magnitude, general specificity and character of the HIV-specific CD8+ T cell response changed little over the study period. While antiretroviral suppression of HIV in chronic infection reduces HIV-specific CD8+ T cell response magnitude in the short term, it had no significant effect on response character over periods up to 9 years.

A Fourth Scale Sensitive to the Magnetic Field; Intermolecular Frequency Symmetry in a Specific Interaction between Protein and Low-Molecular Compound

We found a new method that a specific interaction between prion, i.e., high-molecular compound, and Cp-60, i.e., low-molecular one, could be successfully elucidated with intermolecular frequency symmetry (IFS). To accomplish this, the former sequence is analyzed with a sequence Fourier analysis used average nuclear (N) resonant frequency scale as a fourth one, and the latter structure with a ¹³C-NMR software. Further, such the symmetry could be observed in a specific interaction between a segment of human immunodeficiency virus (HIV)gag and PA-457 or between 1918 neuraminidase and peramivir. Therefore, the IFS rule seems to be evolutionarily conserved as a necessary condition even in a specific protein-organic compound interaction.

Influence of HIV-1 specific T cell response on the progression of HIV-1 infection

Objective To discuss the influence of HIV-1 specific T cell on disease progression of HIV-1 infection.Methods Thirty-seven patients with HIV-1 infection were recruited from a cohort study and their HIV-1 specific T cell responses were analyzed using overlapping peptides and ELISPOT assay.Resuits 83.78% (31/37) of candidates with HIV-1 infection responded to at least one peptide (magnitude was above 50 SFU/106 PBMCs).Peptides from HIV-1 subtype B proteins could elicit specific T cell responses in HIV-1 infections,and amongst them,the responses to HIV-1 Gag were the highest,not only in the frequencies of recognition,hut also in the magnitude of response (F=17.969,P<0.001).In addition,HIV-1 patients with different progression of disease expressed different T cell responses.The magnitude of response elicited by HIV-1 Gag protein in the patients with clinical asymptomatic stage was much higher than that in the patients with AIDS stage,although the responses to whole genome peptides between these two groups were similar.Conclusion The specific T cell responses elicited by different HIV-1 proteins are different,and the responses to HIV-1 Gag protein may play the most important role in the progression of HIV-1 infection. Key words: Human immunodeficiency virus-1 (HIV-1); Cellular immunology; Overlapping peptides; Enzyme-linked immunospot assay (ELISPOT)

Targeting of Murine Leukemia Virus Gag to the Plasma Membrane Is Mediated by PI(4,5)P 2 /PS and a Polybasic Region in the Matrix

Membrane targeting of the human immunodeficiency virus Gag proteins is dependent on phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] located in the plasma membrane. In order to determine if evolutionarily distant retroviral Gag proteins are targeted by a similar mechanism, we generated mutants of the matrix (MA) domain of murine leukemia virus (MuLV) Gag, examined their binding to membrane models in vitro, and analyzed their phenotypes in cell culture. In vitro, we showed that MA bound all the phosphatidylinositol phosphates with significant affinity but displayed a strong specificity for PI(4,5)P(2) only if enhanced by phosphatidylserine. Mutations in the polybasic region in MA dramatically reduced this affinity. In cells, virus production was strongly impaired by PI(4,5)P(2) depletion under conditions of 5ptaseIV overexpression, and mutations in the MA polybasic region altered Gag localization, membrane binding, and virion production. Our results suggest that the N-terminal polybasic cluster of MA is essential for Gag targeting to the plasma membrane. The binding of the MA domain to PI(4,5)P(2) appears to be a conserved feature among retroviruses despite the fact that the MuLV-MA domain is structurally different from that of human immunodeficiency virus types 1 and 2 and lacks a readily identifiable PI(4,5)P(2) binding cleft.

In Vivo Study on Vertical Transmission of the HIV-1 gag Gene via Mouse Oocytes

Acquired immunodeficiency syndrome (AIDS) is a major public health problem worldwide. This study was performed to explore the feasibility of vertical transmission of human immunodeficiency virus-1 (HIV-1) gag gene via oocyte. The recombinant plasmid (pIRES2-EGFP-gag) was injected into mouse ovaries to transfect germ cells. Induction of superovulation and then animal mating were performed to collect oocytes and two-cell embryos. Positive FISH signals for HIV-1 gag DNA were detected in the nuclei of oocytes and embryos, and in chromosomes of mature oocytes, indicated integration of the gene into the oocyte genome and gene replication in the embryo. HIV-1 gag cDNA positive bands detected by RT-PCR in oocytes and embryos indicated successful gene transcription, while positive immunofluorescence signals for HIV-1 gag protein indicated successful translation in both oocytes and embryos. The HIV-1 gag gene was transmitted vertically to the next generation via oocytes and it retained its function in replication, transcription and translation following at least one mitotic division in embryos.

SiRNA Knockdown of PD-L1 and PD-L2 in Monocyte-Derived Dendritic Cells only Modestly Improves Proliferative Responses to Gag by CD8+ T Cells from HIV-1-Infected Individuals

Due to their capacity to elicit and regulate immunity, dendritic cells (DCs) are important targets to improve vaccination. Knowing that programmed death-1 (PD-1) high virus-specific T cells become functionally exhausted during chronic exposure to human immunodeficiency virus-1 (HIV-1), the development of a therapeutic DC-based HIV-1 vaccine might include strategies that downregulate PD-L1 and PD-L2 counter-receptors. After showing that monocyte-derived DCs rapidly upregulated PD-L1 and PD-L2 expression upon maturation with a variety of stimuli, e.g., Toll-like receptor ligands and cytokines, we determined that PD-L1 and PD-L2 expression could be knocked down by electroporation of a single small interfering RNA (siRNA) sequence twice at the monocyte and immature stages of DC development. This knockdown approached completion and was specific and lasting for several days. We then added the PD-L1 and PD-L2 silenced monocyte-derived DCs to peripheral blood mononuclear cells from HIV-1-infected individuals along with pools of 15-mer HIV-1 Gag p24 peptides. However, in cultures from six patients, there was only a modest enhancing effect of PD-L1 and PD-L2 silencing on CD8(+) T cell proliferative responses to the DCs. These findings suggest that, in monocyte-derived DCs, additional strategies than PD-L1 or PD-L2 blockade will be needed to improve the function of PD-1 high T cells.

Packaging limits and stability of HIV-1 sequences in a coxsackievirus B vector

Enteroviruses elicit protective mucosal immune responses that could be harnessed as part of a strategy to prevent sexual transmission of the human immunodeficiency virus-1 (HIV-1). We report the construction of replication-competent recombinant vectors of coxsackievirus B3 (CVB3) that express one or more portions of the HIV-1 Gag protein. Vectors containing the capsid domain of Gag were initially genetically unstable with protein expression lost after brief passage in tissue culture. Codon modification to increase the G/C content of the HIV-1 capsid sequence resulted in enhanced genetic stability of CVB3 vectors during in vitro passage. Cells infected with a vector expressing the matrix (MA) subunit of the HIV-1 Gag protein were susceptible to lysis by CD8 T cell clones specific for the SL9 epitope found within MA. These studies suggest that CVB3 vectors may be useful as vaccine vector candidates, if hurdles in class I antigen presentation and stability can be overcome.

Human Immunodeficiency Virus (HIV) Antigens and RNA in HIV-Seronegative Women with Cervical Intraepithelial Neoplasia

While investigating whether proteins retrieved by cervicovaginal lavages (CVL) from women with cervical intraepithelial neoplasia (CIN) might correlate with risk of progression to invasive cervical cancer, we unexpectedly identified HIV gag and env glycoprotein in CVL from women with HIV-negative serology. HIV antigens were consistently identified by mass spectrometry (MS) in CVL from 4 women but were absent in CVL from the remaining 16 women. HIV serologies of all 20 patients were negative for both HIV-1 and HIV-2 antibodies. To validate the unexpected MS findings we performed Western blot (WB) and immunoaffinity chromatography (IC) analysis of CVL for HIV proteins, viral load assays of paired CVL and blood samples, and immunohistochemical HIV p24 expression in cervical biopsy specimens. WB analysis of CVL for prostate-specific antigen (PSA) was performed to exclude semen contamination as the source of HIV proteins. WB and IC results demonstrated the presence of HIV-1 gp41 and p24 antigens in four CVL that were identified by MS to have the HIV proteins. Despite negative serology, HIV RNA in CVL and HIV p24 in cervix biopsies were detected in patients with HIV antigen-positive CVL. HIV p24-positive CVL were PSA negative. All 20 subjects remained HIV seronegative throughout the study. Women with HIV proteins and RNA were comparatively older. Our findings suggest that CVL HIV proteins in women with CIN could be markers for unrecognized HIV exposure or subclinical infection. Proteomic screening of cervical secretions may be useful in identifying seronegative women exposed to HIV and/or at risk for AIDS.

High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts.

Plants have been recognized as a promising production platform for recombinant pharmaceutical proteins. The human immunodeficiency virus Gag (Pr55(gag)) structural polyprotein precursor is a prime candidate for developing a HIV-1 vaccine, but, so far, has been expressed at very low level in plants. The aim of this study was to investigate factors potentially involved in Pr55(gag) expression and increase protein yield in plant cells. In transient expression experiments in various subcellular compartments, the native Pr55(gag) sequence could be expressed only in the chloroplast. Experiments with truncated subunits suggested a negative role of the 5'-end on the expression of the full gene in the cytosol. Stable transgenic plants were produced in tobacco by Agrobacterium-mediated nuclear transformation with protein targeted to plastids, and biolistic-mediated plastid transformation. Compared to the nuclear genome, the integration and expression of the gag transgene in the plastome resulted in significantly higher protein accumulation levels (up to 7-8% TSP, equivalent to 312-363 mg/kg FW). In transplastomic plants, a 25-fold higher protein accumulation was obtained by translationally fusing the Pr55(gag) polyprotein to the N-terminus of the plastid photosynthetic RbcL protein. In chloroplasts, the Pr55(gag) polyprotein was processed in a pattern similar to that achieved by the viral protease, the processing being more extended in older leaves of mature plants. The Gag proteins produced in transgenic plastids were able to assemble into particles resembling VLPs produced in baculovirus/insect cells and E. coli systems. These results indicate that plastid transformation is a promising tool for HIV antigen manufacturing in plant cells.

Identification and characterization of 2 HIV-1 Gag immunodominant epitopes restricted by Asian HLA allele HLA-B*4801

HLA-B*4801 is frequently found in Asian populations but rarely in Caucasian or African populations. Although HLA-B*4801–restricted human immunodeficiency virus–1 (HIV-1) epitopes would be useful for acquired immune deficiency syndrome (AIDS) vaccine development in Asia, they have not been reported so far. In the present study, we sought to identify HLA-B*4801–restricted HIV-1 epitopes by using 17-mer overlapping peptides derived from HIV-1 Gag, Pol, and Nef as well as 8- to11-mer truncated peptides, and thereby identified two HLA-B*4801–restricted Gag epitopes. These epitope-specific CD8 + T cells strongly responded to HIV-1–infected cells expressing HLA-B*4801, confirming that these Gag epitopes were endogenously presented by HLA-B*4801. These epitope-specific CD8 + T cells were elicited in five of the seven tested chronically HIV-1–infected individuals with HLA-B*4801, suggesting them to be immunodominant epitopes. These epitopes will be useful for the studies of AIDS immunopathogenesis and the development of an HIV-1 vaccine in Asia.