Human Immunodeficiency Virus Type 1 Gag P24 Research Articles

Development of a Live Recombinant BCG Expressing Human Immunodeficiency Virus Type 1 (HIV-1) Gag Using a pMyong2 Vector System: Potential Use As a Novel HIV-1 Vaccine.

Even though the rate of new human immunodeficiency virus type 1 (HIV-1) infections is gradually decreasing worldwide, an effective preventive vaccine for HIV-1 is still urgently needed. The recombinant Mycobacterium bovis BCG (rBCG) is promising for the development of an HIV-1 vaccine. Recently, we showed that a recombinant Mycobacterium smegmatis expressing HIV-1 gag in a pMyong2 vector system (rSmeg-pMyong2-p24) increased the efficacy of a vaccine against HIV-1 in mice. Here, we evaluated the potential of an rBCG expressing HIV-1 p24 antigen Gag in pMyong2 (rBCG-pMyong2-p24) in a vaccine application for HIV-1 infection. We found that rBCG-pMyong2-p24 elicited an enhanced HIV-1 p24 Gag expression in rBCG and infected antigen-presenting cells. We also found that compared to rBCG-pAL-p24 in a pAL5000 derived vector system, rBCG-pMyong2-p24 elicited enhanced p24-specific immune responses in vaccinated mice as evidenced by higher levels of HIV-1 Gag-specific CD4 and CD8 T lymphocyte proliferation, gamma interferon ELISPOT cell induction, antibody production, and cytotoxic T lymphocytes (CTL) responses. Furthermore, rBCG-pMyong2-p24 showed a higher level of p24-specific Ab production than rSmeg-pMyong2-p24 in the same pMyong2 vector system. In conclusion, our data indicated that a live recombinant BCG expressing HIV-1 Gag using a pMyong2 vector system, rBCG-pMyong2-p24 elicited an enhanced immune response against HIV-1 infections in a mouse model system. So, rBCG-pMyong2-p24 may have the potential as a prime vaccine in a heterologous prime-boost vaccine strategy for HIV-1 infection.

Broadening of the T-cell repertoire to HIV-1 Gag p24 by vaccination of HLA-A2/DR transgenic mice with overlapping peptides in the CAF05 adjuvant.

Induction of broad T-cell immune responses is regarded as critical for vaccines against the human immunodeficiency virus type 1 (HIV-1) which exhibit high diversity and, therefore, focus has been on inducing cytotoxic CD8 T-cell responses against the more conserved parts of the virus, such as the Gag protein. Herein, we have used the p24 protein which contains a range of conserved T-cell epitopes. We demonstrate that a vaccine of HIV-1 subtype B consensus group-specific antigen (Gag) p24 protein with the CD8-inducing liposomal cationic adjuvant formulation (CAF) 05, induces both CD4 and CD8 T-cell responses in CB6F1 mice. The adjuvanted vaccine also induced functional antigen-specific cytotoxicity in vivo. Furthermore, we found that when fragmenting the Gag p24 protein into overlapping Gag p24 peptides, a broader T-cell epitope specificity was induced in the humanized human leukocyte antigen (HLA)-A2/DR-transgenic mouse model. Thus, combining overlapping Gag p24 peptides with CAF05 appears to be a promising and simple strategy for inducing broader T-cell responses to multiple conserved epitopes which will be relevant for both prophylactic and therapeutic HIV-1 vaccines.

HIV-1 gag specific exosome-targeted CD8+ T cell based vaccine stimulates specific CTL responses leading to long-term immunity (P4493)

Abstract The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutics for human immunodeficiency virus type-1 (HIV-1)-infected patients with dysfunctional dendritic cells (DCs) and CD4+T cell deficiency. We have recently demonstrated that Con A stimulated non-specific CD8+T cells could uptake OVA pulsed DC-derived exosome (EXO), act as CD8+Tc antigen presenting cells (Th-APCs), and capable of stimulating DC- and CD4+T cell-independent OVA-specific CTL responses leading to long-term immunity. This formed the new concept novel EXO-targeted CD8+T cell vaccine. The extreme conservation and intolerance of escape variants in HIV-1 Gag p24 make it a potential target for vaccine design. In this study, we constructed adenoviral vector AdV-gag and expression vector pEGFP-gag both expressing Gag. We generated Gag specific EXO-targeted T (Gag-Texo) cell vaccine by using C57BL/6 mouse CD8+ ConA T cells with uptake of AdV-gag transfected bone marrow DC (DCgag) released EXOgag. We then generated BL6-10gag cell lines by transfection of B16 melanoma cell line (BL6-10) with pEGFP-gag. We demonstrated that Gag-Texo vaccine is capable of stimulating efficient Gag-specific CD8+ CTL responses in vivo, leading to antitumor immunity against Gag expressing B16 melanoma (BL6-10 gag) in animal studies. Therefore, this novel HIV-1-specific EXO-targeted Gag-Texo vaccine may be useful in induction of efficient CTL responses in AIDS patients.

High-throughput Screening Uncovers a Compound That Activates Latent HIV-1 and Acts Cooperatively with a Histone Deacetylase (HDAC) Inhibitor

Current antiretroviral therapy (ART) provides potent suppression of HIV-1 replication. However, ART does not target latent viral reservoirs, so persistent infection remains a challenge. Small molecules with pharmacological properties that allow them to reach and activate viral reservoirs could potentially be utilized to eliminate the latent arm of the infection when used in combination with ART. Here we describe a cell-based system modeling HIV-1 latency that was utilized in a high-throughput screen to identify small molecule antagonists of HIV-1 latency. A more detailed analysis is provided for one of the hit compounds, antiviral 6 (AV6), which required nuclear factor of activated T cells for early mRNA expression while exhibiting RNA-stabilizing activity. It was found that AV6 reproducibly activated latent provirus from different lymphocyte-based clonal cell lines as well as from latently infected primary resting CD4(+) T cells without causing general T cell proliferation or activation. Moreover, AV6 complemented the latency antagonist activity of a previously described histone deacetylase (HDAC) inhibitor. This is a proof of concept showing that a high-throughput screen employing a cell-based model of HIV-1 latency can be utilized to identify new classes of compounds that can be used in concert with other persistent antagonists with the aim of viral clearance.

112. Strong Transgene-Specific Immune Responses Can Be Elicited with Novel Adeno-Associated Virus Vectors in Mice

Vectors based on recombinant adeno-associated virus (rAAV) have been widely appreciated for gene therapy application, in which the sustained expression of self-genes has been successfully achieved in a variety of animal models. However, the potential of rAAV vectors as vaccine carriers has only recently been recognized. We have isolated several novel AAV serotypes from primates i.e AAVs 7, 8, and 9. Therefore, it is of our interest to develop potential vaccines for human immunodeficiency virus type 1 (HIV-1) based on rAAV vectors of these new serotypes. We have constructed a panel of rAAV vectors based on different AAV serotypes (including AAV2/1, AAV2, AAV2/5, AAV2/7, AAV2/8, and AAV2/9), which all encoded HIV-1 gag p17 and p24 (designated as gagshort). BALB/c mice were i.m. immunized with this panel of AAVHIVgagshort vectors at the dose of E11 genome copies/mouse. 2, 3, and 4 weeks after immunization, splenocytes were harvested and stimulated with the H-2d restricted gag-specific CD8 T-cell epitope in intracellular IFN-g staining. Overall, AAV2/7 and AAV2/8 vaccine vectors elicited the highest levels of gag-specific CD8 T-cell responses; while AAV2/1 and AAV2/9 vaccine vectors were able to induce intermediate levels of gag-specific CD8 T-cell responses. Gag-specific CD8 T-cell responses induced with AAV2/5 and AAV2/2 vaccine vectors were barely detectable. In addition, serum samples from immunized mice were collected and total IgG responses to gag p24 were measured by ELISA. Overall, AAV2/7 and AAV2/9 vaccine vectors yielded the strongest IgG responses against p24; while AAV2/8, AAV2/1, and AAV2/5 vaccine vectors produced the intermediate IgG responses against p24. Surprisingly, there was no detectable p24-specific IgG response in serum samples from mice immunized with AAV2/2HIVgagshort vector, even though this vector yielded very high level of p24 expression in transduced cells in vitro. Interestingly, different patterns of p24-specific IgG1 vs IgG2a responses were observed in serum samples from mice immunized with HIVgagshort vaccine vectors based on different AAV serotypes. Furthermore, significant inflammation and infiltration in the mouse muscles injected with AAV2/7 and AAV2/8 vaccine vectors were observed at 4 weeks after i.m. injection by H/E staining. Taken together, these data suggested that strong transgene-specific immune responses can be elicited with novel rAAV vectors in mice. The immunogenicity of these vectors based on novel AAV serotypes will be examined in nonhuman primates.

Human Immunodeficiency Virus Type 1 (HIV-1)-Specific CD4 + T Cells That Proliferate In Vitro Detected in Samples from Most Viremic Subjects and Inversely Associated with Plasma HIV-1 Levels

Diminished in vitro proliferation of human immunodeficiency virus type 1 (HIV-1)-specific CD4+T cells has been associated with HIV-1 viremia and declining CD4+ T-cell counts during chronic infection. To better understand this phenomenon, we examined whether HIV-1 Gag p24 antigen-induced CD4+ T-cell proliferation might recover in vitro in a group of subjects with chronic HIV-1 viremia and no history of antiretroviral therapy (ART). We found that depletion of CD8+ cells from peripheral blood mononuclear cells (PBMC) before antigen stimulation was associated with a 6.5-fold increase in the median p24-induced CD4+ T-cell proliferative response and a 57% increase in the number of subjects with positive responses. These p24-induced CD4+ T-cell proliferative responses from CD8-depleted PBMC were associated with expansion of the numbers of p24-specific, gamma interferon (IFN-gamma)-producing CD4+ T cells. Among the 20 viremic, treatment-naive subjects studied, the only 5 subjects lacking proliferation-competent, p24-specific CD4+ T-cell responses from CD8-depleted PBMC showed plasma HIV-1 RNA levels > 100,000 copies/ml. Furthermore, both the magnitude of p24-induced CD4+ T-cell proliferative responses from CD8-depleted PBMC and the frequency of p24-specific, IFN-gamma-producing CD4+ T cells expanded from CD8-depleted PBMC were associated inversely with plasma HIV-1 RNA levels. Therefore, proliferation-competent, HIV-1-specific CD4+ T cells that might help control HIV-1 disease may persist during chronic, progressive HIV-1 disease except at very high levels of in vivo HIV-1 replication.

Studies on the generation and maintenance of mucosal cytotoxic T lymphocytes against human immunodeficiency virus type 1 Gag in mice.

Aspects of the generation and maintenance of mucosal immunity against human immunodeficiency virus type 1 (HIV-1) were examined. Mice were immunized either intranasally or intrarectally with recombinant HIV-1 Gag p24 protein plus cholera toxin. Nasal immunization generated strong nasal IgA responses but low vaginal IgA, whereas rectal immunization yielded good vaginal IgA responses but poor nasal responses. Nasal immunization resulted in strong Gag-specific cytotoxic T lymphocyte (CTL) activity in nasal-associated lymphoid tissue (NALT), posterior cervical lymph nodes (pCLNs), and the spleen, but not in mesenteric lymph nodes (MLNs). Rectal immunization induced weak Gag-specific CTLs in the MLNs only, indicating distinct compartmentalization of the upper and lower mucosa. Combining nasal and rectal immunizations overcame their respective deficiencies. CTL memory after the third nasal immunization was found to persist for up to 6 months in the draining pCLNs, but was gradually lost from the NALT induction site. Analysis of the T cell receptor Vbeta usage of Gag-specific CD8(+) T cells in lymphoid tissues of intranasally immunized mice indicated that the memory CTLs in the pCLNs are generated from a few clones in NALT. The memory CTL clones also appear to be poor killers whereas the NALT clones from which the pCLN clones appear to originate are potent killers. Our results support the view that CTL activity is determined by the level and duration of antigen stimulation and that in NALT, CTLs develop as effector memory T cells with high avidity, whereas the pCLNs sequester the memory T cells with low avidity but longer survival.

Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load.

Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1); however, the breadth of these responses at the single-epitope level has not been comprehensively assessed. We therefore screened peripheral blood mononuclear cells (PBMC) from 57 individuals at different stages of HIV-1 infection for virus-specific T-cell responses using a matrix of 504 overlapping peptides spanning all expressed HIV-1 proteins in a gamma interferon-enzyme-linked immunospot (Elispot) assay. HIV-1-specific T-cell responses were detectable in all study subjects, with a median of 14 individual epitopic regions targeted per person (range, 2 to 42), and all 14 HIV-1 protein subunits were recognized. HIV-1 p24-Gag and Nef contained the highest epitope density and were also the most frequently recognized HIV-1 proteins. The total magnitude of the HIV-1-specific response ranged from 280 to 25,860 spot-forming cells (SFC)/10(6) PBMC (median, 4,245) among all study participants. However, the number of epitopic regions targeted, the protein subunits recognized, and the total magnitude of HIV-1-specific responses varied significantly among the tested individuals, with the strongest and broadest responses detectable in individuals with untreated chronic HIV-1 infection. Neither the breadth nor the magnitude of the total HIV-1-specific CD8+-T-cell responses correlated with plasma viral load. We conclude that a peptide matrix-based Elispot assay allows for rapid, sensitive, specific, and efficient assessment of cellular immune responses directed against the entire expressed HIV-1 genome. These data also suggest that the impact of T-cell responses on control of viral replication cannot be explained by the mere quantification of the magnitude and breadth of the CD8+-T-cell response, even if a comprehensive pan-genome screening approach is applied.

Clustering patterns of cytotoxic T-lymphocyte epitopes in human immunodeficiency virus type 1 (HIV-1) proteins reveal imprints of immune evasion on HIV-1 global variation.

The human cytotoxic T-lymphocyte (CTL) response to human immunodeficiency virus type 1 (HIV-1) has been intensely studied, and hundreds of CTL epitopes have been experimentally defined, published, and compiled in the HIV Molecular Immunology Database. Maps of CTL epitopes on HIV-1 protein sequences reveal that defined epitopes tend to cluster. Here we integrate the global sequence and immunology databases to systematically explore the relationship between HIV-1 amino acid sequences and CTL epitope distributions. CTL responses to five HIV-1 proteins, Gag p17, Gag p24, reverse transcriptase (RT), Env, and Nef, have been particularly well characterized in the literature to date. Through comparing CTL epitope distributions in these five proteins to global protein sequence alignments, we identified distinct characteristics of HIV amino acid sequences that correlate with CTL epitope localization. First, experimentally defined HIV CTL epitopes are concentrated in relatively conserved regions. Second, the highly variable regions that lack epitopes bear cumulative evidence of past immune escape that may make them relatively refractive to CTLs: a paucity of predicted proteasome processing sites and an enrichment for amino acids that do not serve as C-terminal anchor residues. Finally, CTL epitopes are more highly concentrated in alpha-helical regions of proteins. Based on amino acid sequence characteristics, in a blinded fashion, we predicted regions in HIV regulatory and accessory proteins that would be likely to contain CTL epitopes; these predictions were then validated by comparison to new sets of experimentally defined epitopes in HIV-1 Rev, Tat, Vif, and Vpr.

Cytotoxic T lymphocytes recognize structurally diverse, clade-specific and cross-reactive peptides in human immunodeficiency virus type-1 gag through HLA-B53

Human immunodeficiency virus type-1 (HIV-1) cytotoxic T lymphocyte (CTL) epitopes have largely been defined in Caucasian populations infected with clade B virus. Identification of potentially protective CTL epitopes in non-B clade-infected African subjects is important for vaccine development. In a study of CTL responses in clade A-infected Gambians, using cytotoxicity, interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISpot) and HLA-B53-peptide tetramer assays, we identified three HLA-B53-restricted epitopes in HIV-1 gag p24. CTL specific for an epitope in a highly immunogenic region of the p24 protein showed no cross-reactivity to other HIV-1 clades. Two of the epitopes would not have been predicted from the peptide-binding motif due to the absence of a proline anchor at position 2. Structural analysis of HLA-B53 and its relative, HLA B35, enabled us to re-define the peptide-binding motif to include other P2 anchors. These results demonstrate the value of combined immunological and structural analyses in defining novel CTL epitopes and have implications for HIV-1 vaccine design.

Inhibition of the human chemokine receptor CXCR4 by antisense phosphorothioate oligodeoxyribonucleotides

The CXC chemokine receptor CXCR4/fusion, a major coreceptor for the T-cell line T-tropic (X4) HIV-1 virus, plays a critical role in T-tropic virus fusion and entry into permissive cells. In the present study, we describe the effects of an antisense phosphorothioate oligodeoxyribonucleotide (anti-S-ODN) on the inhibition of CXCR4 gene expression in X4 HIV-1 infected HeLa-CD4 cells, to find more efficacious therapeutic possibilities for human immunodeficiency virus type 1 (HIV-1) infection. The naked antisense phosphorothioate oligodeoxyribonucleotide (anti-S-ODN-1), containing the AUG initiation codon at the center of the oligodeoxyribonucleotide, showed a slightly higher inhibitory effect on HIV-1 gag p24 production among all sequences tested. We also examined the concomitant use of a basic peptide transfection reagent, nucleosomal histone proteins (RNP), for the delivery of the anti-S-ODN-1. The anti-S-ODN-1 encapsulated with RNP had higher inhibitory effects on p24 products than the naked anti-S-ODN-1. When the anti-S-ODN-1 encapsulated with RNP was incubated with HeLa-CD4 cells, the surface levels of this chemokine receptor showed high suppression, indicating sequence-specific inhibition. The activities of unmodified oligodeoxyribonucleotide are effectively enhanced by using a basic peptide, RNP.

Distribution and Quantification of Human Immunodeficiency Virus Type 1, Strain JC499, Proviral DNA in Tissues from an Infected Chimpanzee

Data are accumulating to show that the natural history of human immunodeficiency virus type 1 (HIV-1) in chimpanzees closely reproduces that in humans and is influenced by biologic properties of the infecting HIV-1 strain. To determine the distribution and relative amounts of HIV-1, proviral DNA in multiple tissues from a chimpanzee euthanized because of an abdominal tumor and kidney failure was quantified by nested PCR limiting-dilution assays. At death, 21 months after infection with HIV-1JC499, this animal had a CD4+ T-cell count of 268 and 1.7 × 105 copies of virion RNA/ml of plasma. The highest proviral burdens were in peripheral lymph nodes and blood, followed by lung, colon, and spleen; values ranged from 130 to 3350 proviral copies/μg DNA (equivalent to DNA in 150,000 cells). The lowest levels of virus were in the spinal cord, brain, and cecum (0.3 to 2.5 copies/μg DNA), with all other tissues harboring intermediate levels (6.8 to 114 copies/μg DNA). Viral burdens in all tissues were comparable to or greater than those reported for HIV-1-infected humans in all stages of disease. Immunohistochemistry for HIV-1 p24 Gag antigen revealed (i) trapping of HIV-1 on follicular dendritic cells in lymph node germinal centers and (ii) virus in the brain, where it was localized primarily to capillary endothelial cells in the cerebral cortex. Analysis of the genetic diversity of the Env V3 loop in tissues indicated that there was no apparent compartmentalization of HIV-1 variants. Of interest, in 83 of 94 (88.3%) clones sequenced, the unique GYGR motif at the tip of the V3 loop of HIV-1JC499 had reverted to the more common GPGR. The results support the conclusion that HIV-1 has the potential to maintain high viral burdens in chimpanzees and to disseminate to most organs, including the central nervous system. The use of the chimpanzee model with HIV-1JC499 (or related strains) in vaccine efficacy studies should prove valuable, especially when assessing protection against disease. Furthermore, comparison of both replicative properties of HIV-1JC499 with SIVcpz strains and immune responses of chimpanzees infected with these viruses might provide new information about HIV pathogenesis.

Hyperviscosity syndrome secondary to a myeloma‐associated IgG1κ paraprotein strongly reactive against the HIV‐1 p24 gag antigen

Hyperviscosity syndrome secondary to hypergammaglobulinemia is a rare and potentially fatal complication in patients with human immunodeficiency virus type-1 (HIV-1) infection. We studied an HIV-1-positive patient with symptomatic hyperviscosity attributable to IgG1κ multiple myeloma. The patient initially responded to plasmapheresis and was subsequently treated with cytotoxic immunosuppressive chemotherapy. The patient remained asymptomatic during a 3-year follow-up period. The monoclonal IgG1κ gammopathy evolved to a biclonal variant of the same subtype with an expansion of marrow plasma cell population. Western blot analysis demonstrated that this myeloma-associated paraprotein was strongly reactive against the HIV-1 p24 gag antigen. Am. J. Hematol. 64:210–213, 2000. © 2000 Wiley-Liss, Inc.

Microparticles in MF59, a potent adjuvant combination for a recombinant protein vaccine against HIV-1

Novel adjuvant formulations involving PLG microparticles with entrapped recombinant protein antigens (env gp120 and p24 gag) from human immunodeficiency virus type-1 (HIV-1), dispersed in the emulsion adjuvant MF59 were evaluated as potential HIV-1 vaccine candidates in mice and baboons. In mice, the adjuvant combination induced significantly enhanced antibody responses in comparison to either adjuvant used alone. In addition, the polylactide co-glycolide polymer (PLG) microparticles and MF59 combination induced CTL activity against HIV-1 p24 gag. In baboons, the adjuvant combination induced significantly enhanced antibody titers after a single dose of gp120, but the responses were comparable to gp120 in MF59 alone after boosting. Both MF59+gp120 alone and PLG/gp120 in MF59 induced neutralizing antibodies against a T cell line-adapted (TCLA) strain and a primary isolate of HIV-1. In contrast to the observations with gp120, immunization in baboons with PLG/p24 in MF59 induced significantly enhanced antibody responses after boosting, in comparison to immunization with MF59 alone+p24.

The life-cycle of human immunodeficiency virus type 1

The life-cycle of human immunodeficiency virus type 1 (HIV-1) has been studied using several techniques including immunoelectron microscopy and cryomicroscopy. The HIV-1 particle consists of an envelope, a core and the region between the core and the envelope (matrix). Virus particles in the extracellular space are observed as having various profiles: a central or an eccentric round electron-dense core, a bar-shaped electron-dense core, and immature doughnut-shaped particle. HIV-1 particles in the hydrated state were observed by high-resolution electron cryomicroscopy to be spherical and the lipid membrane was clearly resolved as a bilayer. Projections around the circumference were seen to be knob-like. The shapes and sizes of the projections, especially the head parts, were found to vary with each projection. HIV-1 cores were isolated with a mixture of Nonidet P40 and glutaraldehyde, and were confirmed to consist of HIV-1 Gag p24 protein by immunogold labelling. On infection, the HIV-1 virus was found to enter the cell in two ways: membrane fusion and endocytosis. After viral entry, no structures resembling virus particles could be seen in the cytoplasm. In the infected cells, positive reactions by immunolabelling suggest that HIV-1 Gag is produced in membrane-bound structures and transported to the cell surface by the cytoskeletons. A crescent electron-dense layer is then formed underneath the cell membrane. Finally, the virus particle is released from the cell surface and found extracellularly to be a complete virus particle with an electron-dense core. However, several cell clones producing defective mature, doughnut-shaped (immature) or teardrop-shaped particles were found to be produced in the extracellular space. In the doughnut-shaped particles, Gag p17 and p24 proteins exist facing each other against an inner electron-dense ring, suggesting that the inner ring consists of a precursor Gag protein showing a defect at the viral proteinase.

Virologic and serologic markers of rapid progression to AIDS after HIV-1 seroconversion.

The association between early virologic and immunologic events after human immunodeficiency virus type 1 (HIV-1) infection and progression of HIV-1 infection to acquired immunodeficiency syndrome (AIDS) was studied among 59 homosexual men with documented time of seroconversion. Epidemiologic factors, such as number of lifetime sexual partners, history of sexually transmitted diseases, and other factors, also were studied. All 17 seroconverters in the cohort who developed AIDS within 3 years (rapid progressors = RPs) were compared with 42 men without AIDS for at least 6 years seroconversion (nonrapid progressors = non-RPs). Plasma levels of HIV-1 RNA, p24 antigen, antibodies to HIV-1 structural genes, beta-2 microglobulin, neopterin, and interferon-alpha were measured at four time points: (a) the last seronegative visit, (b) the first seropositive visit, (c) the visit closest to AIDS (or the corresponding visit for the non-RPs) and (d) 6 years after seroconversion (for non-RPs). Up to seroconversion, the RPs had a significantly higher number of lifetime sexual partners than non-RPs (503 versus 171, respectively). At the first seropositive visit, RPs had significantly higher concentrations of plasma HIV-1 RNA (p < 0.01) and prevalence of p24 antigenemia (p < 0.001) and significantly lower levels of antibodies to the HIV-1 gag proteins p17 and p24 (p < 0.01-0.001) compared with non-RPs. These differences increased during follow-up visits. Antibodies to p66 and gp120 were significantly different only at the visit closet to AIDS (p < 0.001), as were beta-2 microglobulin and interferon alpha. These findings suggest that early virologic-immunologic events after HIV-1 infection may determine the rate of progression to AIDS. Anti-gag immune response may prevent rapid progression of HIV-1 disease and should be considered for future vaccine studies.

An immunovirological study of central nervous system involvement during HIV-1 infection of chimpanzees.

Chimpanzees infected with human immunodeficiency virus type 1 (HIV-1) are used to model acquired immunodeficiency syndrome (AIDS). Since the central nervous system (CNS) is involved in AIDS, we performed an immunovirological study in 18 chimpanzees inoculated up to 87 months prior to the study (mean, 45 months) with HIV-1 and 8 uninfected controls. Serum and cerebrospinal fluid (CSF) IgG and albumin levels of infected chimpanzees never exceeded those of controls. The CSF/serum albumin ratio was elevated in 1 of 18 infected chimpanzees compared to controls; however, all animals had an elevated ratio indicating a more open blood-brain barrier relative to humans. The intrathecal IgG production index was elevated in only 1 of 18 infected chimpanzees compared to controls. Identical serum and CSF IgG bands were found by isoelectric focusing in 2 of 8 controls and in 1 of 18 infected chimpanzees. None of these bands reacted with recombinant HIV-1 p24gag or gp 120env. HIV-1 was isolated from the peripheral blood of 4 of 18 infected chimpanzees but never from the paired CSF samples. Anti-HIV-1 antibody was detected by a enzyme-linked immunosorbent assay in 18 of 18 paired serum and CSF samples and by Western blot in 18 of 18 serum and 13 of 18 CSF samples from infected chimpanzees without a difference in pattern. Polymerase chain reaction analysis on brain tissue of one animal was negative for HIV-1 sequences. Our results demonstrate that, unlike human infection, chimpanzees inoculated with HIV-1 show no evidence of isolatable virus in the CSF and no evidence of intrathecal anti-HIV-1 antibody synthesis up to several years after experimental infection. The lack of CNS involvement may contribute to the delay or suppression of clinical disease in infected chimpanzees.

Comparative humoral responses to human immunodeficiency virus type 1-p24gag linear B-cell epitopes among individuals showing atypical western immunoblotting reactions and implications for diagnosis

Serum specimens from 25 individuals with an isolated human immunodeficiency virus type 1 (HIV-1) core antigen reactivity in a Western immunoblot test were examined for their reactivities with HIV-1 virions, control cellular antigens, HIV-1-Bru p24gag recombinant protein (p24gag), and a panel of 22 p24gag-derived peptides. The results were as follows: (i) serum specimens from eight HIV-1-uninfected subjects did bind to virions but failed to bind to p24gag; (ii) sera from 13 HIV-1-uninfected subjects and from one HIV-2-infected patient reacted with HIV-1 virions and p24gag but failed to bind to any of the peptides expressing major p24gag epitopes, and (iii) 3 serum specimens obtained from one neonate carrying anti-HIV-1 maternal antibody and from two HIV-1-infected subjects who had seroconverted during the study reacted with HIV-1 virions, p24gag, and one or more peptides containing the major p24gag epitopes. Our data suggest that the combination of p24gag and appropriate peptides could be useful for resolution when atypical Western immunoblot results are encountered.

Comparison of trans-dominant inhibitory mutant human immunodeficiency virus type 1 genes expressed by retroviral vectors in human T lymphocytes

trans-Dominant inhibitory mutant versions of the human immunodeficiency virus type 1 (HIV-1) regulatory genes tat and rev have previously been described. We have constructed a series of retroviral vectors to transduce these genes and compare their inhibitory activities. The inhibitory activities were measured with transient transfection assays by using a reporter which expresses an HIV-1 gag-Escherichia coli lacZ fusion protein with strict dependence on coexpression of both tat and rev. Additionally, the vectors were packaged as amphotropic virions and used to stably transduce human CEM T lymphocytes. The transduced CEM cells were challenged with HIV-1, and the effects of the mutant HIV-1 genes were determined by measuring the levels of HIV-1 p24gag produced. A tat gene substituted at amino acid 41 (tatk41a) retained partial trans-activating activity and lacked inhibitory activity. A tat gene with a premature stop codon at amino acid 54 (tat54ter) showed moderate trans-dominant inhibition of the reporter plasmid but failed to significantly inhibit HIV-1 replication. The M10 rev mutant, with a 2-amino-acid substitution, showed strong trans-dominant inhibitory activity both in the reporter plasmid and in the HIV-1 infection assay. The greatest inhibition of HIV-1 growth was seen when M10 was expressed under the transcriptional control of a human cytomegalovirus promoter; slightly less inhibition was achieved when expression of M10 was controlled by the Moloney murine leukemia virus long terminal repeat, and minimal inhibition was seen when the HIV-1 long terminal repeat controlled the M10 gene. These results demonstrate the potential utility of retroviral vectors expressing trans-dominant inhibitory mutant HIV-1 genes for gene therapy approaches to AIDS.

Host cell membrane proteins on human immunodeficiency virus type 1 after in vitro infection of H9 cells and blood mononuclear cells. An immuno-electron microscopic study

Human immunodeficiency virus type 1 (HIV-1)-infected H9 and blood mononuclear cells (MNCs) were studied by immunogold electron microscopy for the presence of HIV-1 gag p24 protein, env gp41 and gp120 proteins, and host cell molecules CD4, CD11a, CD25, CD54, CD63, HLA class I and HLA-DR. Uninfected H9 cells and MNC membranes labelled for CD4, HLA class I and class II, and, at low density, CD11a and CD54; lysosomal structures in the cytoplasm labelled for CD63. The infected cell surface showed immunolabelling for HIV-1 proteins, as did budding particle-like structures. Immunogold labelling of the cell membrane for CD4 was almost non-existent. The level of immunolabelling for CD11a and CD54 on infected cells was greater than that on uninfected cells; this is presumably related to a state of activation during virus synthesis. Budding particle-like structures and free virions in the intercellular space were immunogold-labelled for all host cell markers investigated. This was confirmed by double immunogold labelling using combinations of HIV-1 gag p24 labelling and labelling for the respective host cell molecule. We conclude that virions generated in HIV-1-infected cells concentrate host-derived molecules on their envelope. Also molecules with a prime function in cellular adhesion concentrate on the virion.