Human Immunodeficiency Virus Type 1 P24 Antigen 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.

Development of Monoclonal Antibodies against HIV-1 p24 Protein and Its Application in Colloidal Gold Immunochromatographic Assay for HIV-1 Detection.

Human immunodeficiency virus type 1 (HIV-1) p24 protein is the most abundant viral protein of HIV-1. This protein is secreted in blood serum at high levels during the early stages of HIV-1 infection, making it a biomarker for early diagnosis. In this study, a colloidal gold immunochromatographic assay (GICA) was established for detecting p24 protein using mouse monoclonal antibodies (mAbs). The HIV-1 p24 protein was expressed in E. coli strain BL21 and the purified protein was used to immunize mice. Stable hybridoma cell lines secreting anti-p24 monoclonal antibodies were obtained after ELISA screening and subcloning by limiting dilution. 34 different capture and labeling mAb pairs were selected by a novel antibody-capture indirect sandwich ELISA and then applied in GICA to detect p24 protein. The GICA method has a limit of detection (LOD) of 25 pg/mL and could detect p24 protein in all 10 positive samples obtained from the National Reference of HIV-1 p24 antigen. Out of 153 negative samples tested, 3 false positives results were obtained. The overall specificity of this test was 98.03%. The good sensitivity and specificity of this method make it a suitable alternative to provide a more convenient and efficient tool for early diagnosis of HIV infection.

Antigen-presenting phagocytic cells ingest malaria parasites and increase HIV replication in a tumor necrosis factor α-dependent manner.

Plasmodium falciparum infection induces human immunodeficiency virus (HIV) replication and accelerates a decline in CD4(+) T-cell count. The mechanisms contributing to these interactions have not been fully elucidated. We infected peripheral blood mononuclear cells (PBMCs) with HIV type 1 (HIV-1) and then cocultured them with P. falciparum-infected red blood cells (iRBCs) or uninfected RBCs (uRBCs). Levels of HIV-1 p24 antigen and activation-associated cytokines were measured in culture supernatants. T-cell surface activation was assessed by flow cytometry. It has been reported that iRBCs increase HIV replication, compared with uRBCs; that neutralizing tumor necrosis factor α (TNF-α) abrogates this increase; and that hemozoin enhances HIV production. In this study, we confirmed that TNF-α plays an important role in this interaction. We show that iRBCs increased CD4(+) T-cell expression of HLA-DR(+)/CD38(+) (P = .001), that monocyte/macrophage depletion reduced HIV production by 40%-50% (P < .001), and that hemozoin-laden monocytes/macrophages that were preincubated with iRBCs also stimulated HIV production. iRBCs activate CD4(+) T cells and stimulate HIV replication in a TNF-α-dependent manner following malarial antigen processing by monocytes/macrophages. These results suggest that the persistent elevation of HIV replication during and after acute bouts of P. falciparum malaria may be due, at least in part, to ongoing stimulation of CD4(+) T cells by hemozoin-loaded antigen-presenting cells within lymphoid tissues.

Oral delivery of plant-derived HIV-1 p24 antigen in low doses shows a superior priming effect in mice compared to high doses

During early infection with human immunodeficiency virus type 1 (HIV-1), there is a rapid depletion of CD4+ T-cells in the gut-associated lymphoid tissue (GALT) in the gastrointestinal tract. Therefore, immediate protection at these surfaces is of high priority for the development of an HIV-1 vaccine. Thus, transgenic plants expressing HIV-1 antigens, which are exposed to immune competent cells in the GALT during oral administration, can be interesting as potential vaccine candidates. In the present study, we used two HIV-1 p24 antigen-expressing transgenic plant systems, Arabidopsis thaliana and Daucus carota, in oral immunization experiments. Both transgenic plant systems showed a priming effect in mice and induced humoral immune responses, which could be detected as anti-p24-specific IgG in sera after an intramuscular p24 protein boost. Dose-dependent antigen analyses using transgenic A. thaliana indicated that low p24 antigen doses were superior to high p24 antigen doses.

Oral delivery of transgenic plant-derived HIV-1 p24 antigen in low doses shows a superior priming effect in mice compared to higher doses

During early infection with human immunodeficiency virus type 1 (HIV-1), there is a rapid depletion of CD4+ T-cells in the gut-associated lymphoid tissue (GALT) in the gastrointestinal tract. There ...

Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen

BackgroundHIV-1 p24 antigen is a major viral component of human immunodeficiency virus type 1 (HIV-1) which can be used to identify persons in the early stage of infection and transmission of HIV-1 from infected mothers to infants. The detection of p24 is usually accomplished by using an enzyme-linked immunosorbent assay (ELISA) with low detection sensitivity. Here we report the use of two bio-barcode amplification (BCA) assays combined with polymerase chain reaction (PCR) and gel electrophoresis to quantify HIV-1 p24 antigen.MethodA pair of anti-p24 monoclonal antibodies (mAbs) were used in BCA assays to capture HIV-1 p24 antigen in a sandwich format and allowed for the quantitative measurement of captured p24 using PCR and gel electrophoresis. The first 1 G12 mAb was coated on microplate wells or magnetic microparticles (MMPs) to capture free p24 antigens. Captured p24 in turn captured 1D4 mAb coated gold nanoparticle probes (GNPs) containing double-stranded DNA oligonucleotides. One strand of the oligonucleotides was covalently immobilized whereas the unbound complimentary bio-barcode DNA strand could be released upon heating. The released bio-barcode DNA was amplified by PCR, electrophoresed in agarose gel and quantified.ResultsThe in-house ELISA assay was found to quantify p24 antigen with a limit of detection (LOD) of 1,000 pg/ml and a linear range between 3,000 and 100,000 pg/ml. In contrast, the BCA-based microplate method yielded an LOD of 1 pg/ml and a linear detection range from 1 to 10,000 pg/ml. The BCA-based MMP method yielded an LOD of 0.1 pg/ml and a linear detection range from 0.1 to 1,000 pg/ml.ConclusionsWhen combined with PCR and simple gel electrophoresis, BCA-based microplate and MMPs assays can be used to quantify HIV-1 p24 antigen. These methods are 3–4 orders of magnitude more sensitive than our in-house ELISA-based assay and may provide a useful approach to detect p24 in patients newly infected with HIV.

Nanoparticle‐Based Immunoassays for Sensitive and Early Detection of HIV‐1 Capsid (p24) Antigen

We evaluated the feasibility of using nanoparticle (NP)-based assays for improving detection sensitivity of human immunodeficiency virus type 1 (HIV-1) p24 antigen. One assay that was evaluated is a gold NP-based biobarcode amplification (BCA) assay, which can detect HIV-1 p24 antigen at levels as low as 0.1 pg/mL. The lower limit of detection for an enzyme-linked immunosorbent assay (ELISA) is 10-15 pg/mL. These results demonstrate that the HIV-1 p24 BCA assay offers 100-150-fold enhancement in the detection limit over the traditional colorimetric ELISA. Furthermore, the BCA assay detected HIV-1 infection 3 days earlier than did ELISA in samples from patients who had experienced seroconversion. The other assay that we tested is the europium NP-based immunoassay, which uses europium NPs to replace gold NPs in the BCA assay to further simplify the detection method and decrease the incubation time. For detection of HIV-1 p24, the lower limit of detection for the europium NP-based immunoassay was 0.5 pg/mL. These results indicate that the universal labeling technology based on NPs and its application may provide a rapid and sensitive testing platform for clinical diagnosis and laboratory research.

Glycoprotein 96-Mediated Presentation of Human Immunodeficiency Virus Type 1 (HIV-1)-Specific Human Leukocyte Antigen Class I-Restricted Peptide and Humoral Immune Responses to HIV-1 p24

Viral antigens complexed to heat shock proteins (HSPs) can enhance antiviral immunity. The present study evaluated the immunogenicity of a novel human immunodeficiency virus type 1B' (HIV-1B')-specific, human leukocyte antigen A2 (HLA-A2)-restricted peptide (FLQSRPEPTA, Gag(448-457)) and the cellular immune adjuvant effect of HSP gp96 using the HLA-A2 transgenic mouse model. It was found that gp96 could augment cytotoxic-T-lymphocyte responses specific for the 10-mer peptide of HIV-1B'. This study also evaluated the humoral immune adjuvant effect of HSP gp96 and its N-terminal fragment (N336) and found that immunization of BALB/c mice with a mixture of gp96 or its N-terminal fragment and HIV-1 p24 antigen or with an p24-N336 fusion protein resulted in a significant increase in anti-HIV p24 antibody titer. These results demonstrate the possibility of using gp96 and its N fragment as adjuvants to augment cellular and humoral immune responses against HIV-1 infection.

Selective infection of CD4+ effector memory T lymphocytes leads to preferential depletion of memory T lymphocytes in R5 HIV-1-infected humanized NOD/SCID/IL-2Rγnull mice

To investigate the events leading to the depletion of CD4(+) T lymphocytes during long-term infection of human immunodeficiency virus type 1 (HIV-1), we infected human CD34(+) cells-transplanted NOD/SCID/IL-2Rgamma(null) mice with CXCR4-tropic and CCR5-tropic HIV-1. CXCR4-tropic HIV-1-infected mice were quickly depleted of CD4(+) thymocytes and both CD45RA(+) naïve and CD45RA(-) memory CD4(+) T lymphocytes, while CCR5-tropic HIV-1-infected mice were preferentially depleted of CD45RA(-) memory CD4(+) T lymphocytes. Staining of HIV-1 p24 antigen revealed that CCR5-tropic HIV-1 preferentially infected effector memory T lymphocytes (T(EM)) rather than central memory T lymphocytes. In addition, the majority of p24(+) cells in CCR5-tropic HIV-1-infected mice were activated and in cycling phase. Taken together, our findings indicate that productive infection mainly takes place in the activated T(EM) in cycling phase and further suggest that the predominant infection in T(EM) would lead to the depletion of memory CD4(+) T lymphocytes in CCR5-tropic HIV-1-infected mice.

Single-molecule immunosorbent assay as a tool for human immunodeficiency virus-1 antigen detection

Ultrasensitive detection and quantification of viral antigen with a novel single-molecule immunosorbent assay (SMISA) was achieved. Antigen from human immunodeficiency virus type 1 (HIV-1), the major etiological agent of acquired immune deficiency syndrome, served as the screening target in this study. The target molecule was sandwiched between a polyclonal capture antibody and a monoclonal detector antibody. The capture antibody was covalently immobilized on (3-glycidoxypropyl) trimethoxy silane-modified glass slides. The detector antibody was conjugated with fluorescent Alexa Fluor 532 labeled secondary antibody prior to being used as a probe for the antigen. Imaging was performed with a total internal reflection fluorescence single-molecule detection system. This technique is demonstrated for detecting HIV-1 p24 antigen down to 0.1 pg/mL with a dynamic range of over four orders of magnitude. A Langmuir isotherm fits the molecule count dependence on the target concentration. The target antigen was further tested in 20% human serum, and the results showed that neither sensitivity nor dynamic range was affected by the biological matrix. SMISA is therefore a promising approach for the early diagnosis of viral induced diseases.

Human cytomegalovirus and human immunodeficiency virus type-1 co-infection in human cervical tissue

Human cytomegalovirus (HCMV) and human immunodeficiency virus type-1 (HIV-1) infect the female genital tract. A human cervical explant model was developed to study single and dual infection by these viruses in the genital compartment. An HCMV strain expressing green fluorescent protein, and two clinical HCMV strains produced peak viral DNA copies at 14 to 21 days post-infection. Peak levels of HIV-1 Ba-L p24 antigen occurred at 7 days post-infection. HIV-1 Ba-L appeared to enhance HCMV in co-infected tissues. Singly and dually infected explants produced increased levels of cytokines IL-6, IL-8, and GRO-α in culture supernatants. Immunohistochemical and flow cytometric analysis showed HCMV infection of leukocytes with the phenotype CD45+/CD1a+/CD14+/HLA-DR+ but not stromal or endothelial cells. Cells expressing both GFP and HIV-1 p24 antigen were detected in co-infected tissues. The cervical explants provide an ex vivo human model for examining mechanisms of virus–virus interaction and pathogenesis in clinically relevant tissue.

Interaction of Human Immunodeficiency Virus Type 1 Integrase with Cellular Nuclear Import Receptor Importin 7 and Its Impact on Viral Replication

Similar to all other viruses, human immunodeficiency virus type 1 (HIV-1) depends heavily on cellular factors for its successful replication. In this study we have investigated the interaction of HIV-1 integrase (IN) with several host nuclear import factors using co-immunoprecipitation assays. Our results indicate that IN interacts specifically with host importin 7 (Imp7) in vivo, but does not interact with importin 8 (Imp8) or importin alpha (Rch1). In contrast, another HIV-1 karyophilic protein MAp17, which is capable of binding Rch1, fails to interact with Imp7, suggesting that IN and Map17 may interact with different cellular pathways during HIV-1 replication. Genetic analysis revealed that the C-terminal domain of IN is the region responsible for interaction between IN with Imp7, and an IN mutant (K240A,K244A/R263A,K264A) disrupted the Imp7 binding ability of the protein, indicating that both regions ((235)WKGPAKLLWKG and (262)RRKAK) within the C-terminal domain of IN are required for efficient IN/Imp7 interaction. Using a vesicular stomatitis virus G glycoprotein pseudotyped HIV single-cycle replication system, we showed that the IN/Imp7 interaction-deficient mutant was unable to mediate viral replication and displayed impairment at both viral reverse transcription and nuclear import steps. Moreover, transient knockdown of Imp7 in both HIV-1 producing and target cells resulted in a 2.5-3.5-fold inhibition of HIV infection. Altogether, our results indicate that HIV-1 IN specifically interacts with Imp7, and this viral/cellular protein interaction contributes to efficient HIV-1 infection.

In vitro suppression of human immunodeficiency virus type 1 replication by measles virus.

During the acute phase of measles, human immunodeficiency virus type 1 (HIV-1)-infected children have a transient, but dramatic, decrease in plasma HIV-1 RNA levels (W. J. Moss, J. J. Ryon, M. Monze, F. Cutts, T. C. Quinn, and D. E. Griffin, J. Infect. Dis. 185:1035-1042, 2002). To determine the mechanism(s) by which coinfection with measles virus (MV) decreases HIV-1 replication, we established an in vitro culture system that reproduces this effect. The addition of MV to CCR5- or CXCR4-tropic HIV-1-infected human peripheral blood mononuclear cells (PBMCs) decreased HIV-1 p24 antigen production in a dose-dependent manner. This decrease occurred with the addition of MV before or after HIV-1. The inhibition of HIV-1 p24 antigen production was decreased when UV-inactivated MV or virus-free supernatant fluid from MV-infected PBMCs was used. Inhibition was not due to increased production of chemokines known to block coreceptor usage by HIV-1, a decrease in the percentage of CD4+ T cells, or a decrease in chemokine receptor expression by CD4+ T cells. Viability of PBMCs was decreased only 10 to 20% by MV coinfection; however, lymphocyte proliferation was decreased by 60 to 90% and correlated with decreased production of p24 antigen. These studies showed that an in vitro system of coinfected PBMCs could be used to dissect the mechanism(s) by which MV suppresses HIV-1 replication in coinfected children and suggest that inhibition of lymphocyte proliferation by MV may play a role in the suppression of HIV-1 p24 antigen production.

The Use of Nanoarrays for Highly Sensitive and Selective Detection of Human Immunodeficiency Virus Type 1 in Plasma

Arrays of antibodies with well-defined feature size and spacing are necessary for developing highly sensitive and selective immunoassays to detect macromolecules in complex solutions. Here we report the application of nanometer-scale antibody array-based analysis to determine the presence of the human immunodeficiency virus type 1 (HIV-1) in blood samples. Dip-pen nanolithography (DPN) was used to generate nanoscale patterns of antibodies against the HIV-1 p24 antigen on a gold surface. Feature sizes were less than 100-nanometers, and the activity of the antibody was preserved. HIV-1 p24 antigen in plasma obtained directly from HIV-1-infected patients was hybridized to the antibody array in situ, and the bound protein was hybridized to a gold antibody-functionalized nanoparticle probe for signal enhancement. The nanoarray features in the three-component sandwich assay were confirmed by atomic force microscopy (AFM). Demonstration of measurable amounts of HIV-1 p24 antigen in plasma obtained from men with ...

Increased Affinity and Stability of an Anti-HIV-1 Envelope Immunotoxin by Structure-based Mutagenesis

HIV-infected cells are selectively killed by an immunotoxin in which a truncated form of Pseudomonas exotoxin A is joined to the variable region of a broadly neutralizing antibody (3B3) that recognizes the viral envelope glycoprotein (Env). To improve the efficacy of this molecule, we used three-dimensional structural information and phage selection data to design 23 single and multiple point mutations in the antibody variable region sequences that contact Env. Substituting an aromatic residue for an aspartate in the third complementarity-determining region of V(H) increased the potency of the immunotoxin by approximately 10-fold in a cell-killing assay. Detailed analysis of one such mutant, N31H/Q100eY, revealed both a higher affinity for monomeric and cell surface Env and an increased stability against aggregation compared with the starting immunotoxin. Conversion to a disulfide-linked two-chain format further stabilized the protein. N31H/Q100eY retained the ability to bind to Env from multiple viral isolates, to inhibit Env-mediated cell fusion, and to limit spreading viral infection in peripheral blood mononuclear cells. Such site-directed mutants may increase the utility of immunotoxins for reducing or eradicating persistent HIV-1 infection in humans.

Significant closure of the human immunodeficiency virus type 1 and hepatitis C virus preseroconversion detection windows with a transcription-mediated-amplification-driven assay.

While the present generation of serology-based assays has significantly decreased the number of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections acquired by transfusion, the possibility of infected donations escaping detection still exists. The average seronegative viremic window duration during which immunological assays are unable to detect the virus is estimated to be between 16 and 22 days for HIV-1 and approximately 70 days for HCV. Significant reduction of detection window duration was demonstrated using a nucleic acid amplification assay, the Procleix HIV-1/HCV Assay, which utilizes transcription-mediated amplification technology to simultaneously detect HIV-1 and HCV RNAs. For 26 commercially available HIV-1 seroconversion panels tested, specimens were reactive in the HIV-1/HCV assay at the same time as or earlier than in serological assays. Overall, the HIV-1/HCV assay was able to reduce the detection window duration by an average of 14 days and 6 days compared to tests relying on recognition of HIV-1 antibody and p24 antigen, respectively. For 24 commercially available HCV seroconversion panels tested, the specimens were reactive in the HIV-1/HCV assay at an earlier blood sampling date than in serological assays, reducing the detection window duration by an average of 26 days. Similar results were obtained in testing the HIV-1 and HCV seroconversion panels in the virus-specific HIV-1- and HCV-discriminatory assays, respectively. In conclusion, the HIV-1/HCV assay and corresponding discriminatory assays significantly reduced detection window durations compared to immunoassays.

Expression and immunogenicity of human immunodeficiency virus type 1 Gag expressed by a replication-competent rhabdovirus-based vaccine vector.

A replication-competent rhabdovirus-based vector expressing human immunodeficiency virus type 1 (HIV-1) Gag protein was characterized on human cell lines and analyzed for the induction of a cellular immune response in mice. We previously described a rabies virus (RV) vaccine strain-based vector expressing HIV-1 gp160. The recombinant RV was able to induce strong humoral and cellular immune responses against the HIV-1 envelope protein in mice (M. J. Schnell et al., Proc. Natl. Acad. Sci. USA 97:3544-3549, 2000; J. P. McGettigan et al., J. Virol. 75:4430-4434, 2001). Recent research suggests that the HIV-1 Gag protein is another important target for cell-mediated host immune defense. Here we show that HIV-1 Gag can efficiently be expressed by RV on both human and nonhuman cell lines. Infection of HeLa cells with recombinant RV expressing HIV-1 Gag resulted in efficient expression of HIV-1 precursor protein p55 as indicated by both immunostaining and Western blotting. Moreover, HIV-1 p24 antigen capture enzyme-linked immunosorbent assay and electron microscopy showed efficient release of HIV-1 virus-like particles in addition to bullet-shaped RV particles in the supernatants of the infected cells. To initially screen the immunogenicity of this new vaccine vector, BALB/c mice received a single vaccination with the recombinant RV expressing HIV-1 Gag. Immunized mice developed a vigorous CD8(+) cytotoxic T-lymphocyte response against HIV-1 Gag. In addition, 26.8% of CD8(+) T cells from mice immunized with RV expressing HIV-1 Gag produced gamma interferon after challenge with a recombinant vaccinia virus expressing HIV-1 Gag. These results further confirm and extend the potency of RV-based vectors as a potential HIV-1 vaccine.

U50,488 protection against HIV-1-related neurotoxicity: involvement of quinolinic acid suppression

The pathogenesis of human immunodeficiency virus type 1 (HIV-1) encephalopathy has been associated with multiple factors including the neurotoxin quinolinate (an endogenous N-methyl- d-aspartate [NMDA] receptor ligand) and viral proteins. The κ opioid receptor (KOR) agonist U50,488 recently has been shown to inhibit HIV-1 p24 antigen production in acutely infected microglial cell cultures. Using primary human brain cell cultures in the present study, we found that U50,488 also suppressed in a dose-dependent manner the neurotoxicity mediated by supernatants derived from HIV-1-infected microglia. This neuroprotective effect of U50,488 was blocked by the KOR selective antagonist nor-binaltorphimine. The neurotoxic activity of the supernatants from HIV-1-infected microglia was blocked by the NMDA receptor antagonists 2-amino-5-phosphonovalerate and MK-801. HIV-1 infection of microglial cell cultures induced the release of quinolinate, and U50,488 dose-dependently suppressed quinolinate release by infected microglial cell cultures with a corresponding inhibition of HIV-1 p24 antigen levels. These findings suggest that the kappa opioid ligand U50,488 may have therapeutic potential in HIV-1 encephalopathy by attenuating microglial cell production of the neurotoxin quinolinate and viral proteins.

Inhibition of replication of reactivated human immunodeficiency virus type 1 (HIV-1) in latently infected U1 cells transduced with an HIV-1 long terminal repeat-driven PKR cDNA construct.

Treatment of human immunodeficiency virus type 1 (HIV-1)-infected individuals with highly active antiretroviral therapy has effectively decreased viral load to undetectable levels. However, efforts to eliminate HIV-1 from these individuals have been unsuccessful, due to the presence of stable, latent viral reservoirs in resting and active CD4(+) T lymphocytes and macrophages. These latent populations have become critical targets in the effort to eradicate HIV-1 from infected individuals. The mechanisms of HIV-1 latency have been studied by using the HIV-1-infected promonocytic cell line U1. The interferon-inducible double-stranded RNA-dependent p68 protein kinase (PKR), a key enzyme in the host-mediated antiviral response, is known to be down-regulated during HIV-1 infection. Therefore, in order to evaluate the role of PKR in the inhibition of replication of reactivated HIV-1 in latently infected U1 cells, we have utilized cDNA constructs containing PKR under the transcriptional control of the HIV-1 long terminal repeat. One PKR-transduced clone, U1/106-4:27, inhibited the tumor necrosis factor alpha (TNF-alpha)-induced replication of HIV-1 by 99% compared to control U1 cells as measured by syncytium formation and HIV-1 p24 antigen enzyme-linked immunosorbent assay. Western blot analysis showed an increase in PKR expression through 96 h postinduction in the U1/106-4:27 clone, concomitant with maximal increases in phosphorylation of the alpha subunit of eukaryotic initiation factor 2 and NF-kappaB activity at 72 h postinduction. These results demonstrate that overexpression of PKR can inhibit the replication of reactivated HIV-1 in latently infected cells and confirm the involvement of PKR in the interferon-associated antiviral pathway against HIV-1 infection. Additionally, treatment of the PKR-transduced U1/106-4:27 clone with the protease inhibitor saquinavir (250 nM) completely inhibited TNF-alpha-induced HIV-1 replication.

Effect of immune activation induced by Cryptosporidium parvum whole antigen on in vitro human immunodeficiency virus type 1 infection.

Previous epidemiologic investigations have suggested that persons with AIDS who are infected with Cryptosporidium parvum have a shorter survival time than those with other opportunistic infections. In this study, the effect of immune activation by a crude Cryptosporidium whole antigen on human immunodeficiency virus type 1 (HIV-1) infection was evaluated. Peripheral blood mononuclear cells from healthy persons without HIV-1 infection had increased proliferative and cytokine (interleukin-4, interferon-gamma, and tumor necrosis factor [TNF]-alpha) responses to stimulation with the crude Cryptosporidium whole antigen. This stimulation increased HIV-1 p24 antigen production in in vitro infection by>30-fold. A similar increase in p24 production was also seen when stimulation was done after cells were infected with HIV-1. Neutralization of TNF-alpha reduced Cryptosporidium antigen-induced p24 production by >50%. Results of this study suggest that Cryptosporidium-induced immune activation may be a cofactor in regulating HIV-1 production.