HIV-1 Vaccine-Induced T-Cell Reponses Cluster in Epitope Hotspots that Differ from Those Induced in Natural Infection with HIV-1

Several recent large clinical trials evaluated HIV vaccine candidates that were based on recombinant adenovirus serotype 5 (rAd-5) vectors expressing HIV-derived antigens. These vaccines primarily elicited T-cell responses, which are known to be critical for controlling HIV infection. In the current study, we present a meta-analysis of epitope mapping data from 177 participants in three clinical trials that tested two different HIV vaccines: MRKAd-5 HIV and VRC-HIVAD014-00VP. We characterized the population-level epitope responses in these trials by generating population-based epitope maps, and also designed such maps using a large cohort of 372 naturally infected individuals. We used these maps to address several questions: (1) Are vaccine-induced responses randomly distributed across vaccine inserts, or do they cluster into immunodominant epitope hotspots? (2) Are the immunodominance patterns observed for these two vaccines in three vaccine trials different from one another? (3) Do vaccine-induced hotspots overlap with epitope hotspots induced by chronic natural infection with HIV-1? (4) Do immunodominant hotspots target evolutionarily conserved regions of the HIV genome? (5) Can epitope prediction methods be used to identify these hotspots? We found that vaccine responses clustered into epitope hotspots in all three vaccine trials and some of these hotspots were not observed in chronic natural infection. We also found significant differences between the immunodominance patterns generated in each trial, even comparing two trials that tested the same vaccine in different populations. Some of the vaccine-induced immunodominant hotspots were located in highly variable regions of the HIV genome, and this was more evident for the MRKAd-5 HIV vaccine. Finally, we found that epitope prediction methods can partially predict the location of vaccine-induced epitope hotspots. Our findings have implications for vaccine design and suggest a framework by which different vaccine candidates can be compared in early phases of evaluation.     

Mechanisms of alpha1-antitrypsin inhibition of cellular serine proteases and HIV-1 protease that are essential for HIV-1 morphogenesis

Proprotein processing is essential for HIV infectivity. Cellular trans-Golgi network (TGN) serine proteases (e.g., furin) are required to cleave HIV envelope gp160 to gp120. In addition, HIV protease (PR), an aspartyl protease, cleaves p55(Gag) to p24, etc., in budding virions. alpha1-Antitrypsin (alpha(1)AT) is cleaved by serine proteases, causing a conformational change in alpha(1)AT that sequesters and so inactivates the protease. alpha(1)AT blocks both gp160 and p55 processing, and so is a powerful inhibitor of HIV replication. We hypothesized that alpha(1)AT inhibited gp160 and p55 processing via different mechanisms, and that in both cases, alpha(1)AT bound and was itself cleaved by the proteases whose activities were blocked. alpha(1)AT delivered by SV(AT), a recombinant, Tag-deleted SV40-derived vector, localized to the TGN, co-precipitated with furin, and depleted furin from the TGN. After SV(AT) transduction and HIV challenge, alpha(1)AT was detected in resulting nascent immature HIV-1 virions. alpha(1)AT also blocked incorporation of the enzymatically active dimeric form of PR into HIV virions. Western analysis using recombinant proteins showed that alpha(1)AT directly bound HIV PR, and was cleaved by it. The simultaneous inhibition of two different steps in HIV morphogenesis both increases alpha(1)AT antilentiviral activity and decreases the possibility that HIV mutations will allow escape from inhibition.     

HIV-1 does not provoke alteration of cytokine gene expression in lymphoid tissue after acute infection ex vivo

The cytokine response to invading microorganisms is critical for priming the adaptive immune response. During acute HIV infection, the response is disrupted, but the mechanism is poorly understood. We examined the cytokine response in human lymphoid tissue, acutely infected ex vivo with HIV. Lymphoid tissue was cultured either as blocks or as human lymphocyte aggregate cultures (HLAC) of tonsils and lymph nodes. This approach allowed us to examine the effects of HIV on cytokines using distinct culture techniques. In contrast to HLAC, mock-infected tissue blocks displayed a 50- to 100-fold up-regulation of mRNAs for IL-1beta, -6, and -8 in the first 6 days of culture. Parallel increases were also noted at the protein level in the supernatants. Although IL-1beta, -6, and -8 are known to synergistically enhance HIV replication, peak HIV replication (measured as p24 Ag) was similar in tissue blocks and HLAC. Surprisingly, vigorous HIV replication of CXCR4- and CCR5-tropic HIV strains did not result in characteristic mRNA profiles for IL-1beta, -2, -4, -6, -8, -10, -12, -15, IFN-gamma, TNF-alpha, TGF-beta, and beta-chemokines in tissue blocks or HLAC. The increased expression of IL-1beta, -6, and -8 in tissue blocks may approximate clinical situations with heightened immune activation; neutralization of these cytokines resulted in inhibition of HIV replication, suggesting that these cytokines may contribute to HIV replication in certain clinical settings. These results also indicate that different molecular mechanisms govern HIV replication in tissue blocks and HLAC. Prevention of effective cytokine responses may be an important mechanism that HIV uses during acute infection.     

A hybridoma producing human monoclonal antibody specific for glycoprotein 120 kDa of human immunodeficiency virus (HIV-1

A stable hybridoma producing anti-HIV human monoclonal antibody (HMCA) was generated by fusing CD3-depleted human splenic lymphocytes from an HIV sero-positive donor with the mouse myeloma cell line P3x63AgU1. The resultant hybridoma has been secreting IgG1, lambda chain for over nine months at a rate of 2.5 micrograms/10(6)cells/day. The HMCA shows specific reactivity in ELISA using HIV-infected cell lysates. Immunofluorescence tests have indicated that this HMCA binds specifically to the surface of H9 and C3 HIV/HTLVIIIb infected cells, HIV/N1T infected CEM cells and to MoT cells infected with an HIV clinical isolate. Western blotting revealed recognition of glycoproteins 120 and 160 kDa of HIV by the HMCA. Although this HMCA demonstrated no neutralizing activity, the production of an anti-HIV HMCA specific for glycoprotein 120 kDa indicates the possibility that a neutralizing HMCA can be developed as further fusions with lymph nodes and spleens from HIV positive donors are performed.     

Human immunodeficiency virus type 1 (HIV-1)-induced GRO-alpha production stimulates HIV-1 replication in macrophages and T lymphocytes

We examined the early effects of infection by CCR5-using (R5 human immunodeficiency virus [HIV]) and CXCR4-using (X4 HIV) strains of HIV type 1 (HIV-1) on chemokine production by primary human monocyte-derived macrophages (MDM). While R5 HIV, but not X4 HIV, replicated in MDM, we found that the production of the C-X-C chemokine growth-regulated oncogene alpha (GRO-alpha) was markedly stimulated by X4 HIV and, to a much lesser extent, by R5 HIV. HIV-1 gp120 engagement of CXCR4 initiated the stimulation of GRO-alpha production, an effect blocked by antibodies to CXCR4. GRO-alpha then fed back and stimulated HIV-1 replication in both MDM and lymphocytes, and antibodies that neutralize GRO-alpha or CXCR2 (the receptor for GRO-alpha) markedly reduced viral replication in MDM and peripheral blood mononuclear cells. Therefore, activation of MDM by HIV-1 gp120 engagement of CXCR4 initiates an autocrine-paracrine loop that may be important in disease progression after the emergence of X4 HIV.     

Antibody to adhesion molecule LFA-1 enhances plasma neutralization of human immunodeficiency virus type 1.

We have shown that a monoclonal antibody to the cell surface adhesion molecule LFA-1 (CD18/CD11a) enhances plasma neutralization of a laboratory isolate (HIVMN) and a primary isolate (HIV28R) of human immunodeficiency virus type 1. Human phytohemagglutinin blasts were infected with HIVMN or HIV28R in the presence of plasma pooled from HIV-positive individuals (AIDS plasma) or immunoglobulin G from AIDS plasma alone or combined with a monoclonal antibody (MAb) to LFA-1. While AIDS plasma alone at a dilution of 1:1,250 neutralized HIVMN and HIV28R infection by 15 and 0%, respectively, in the presence of a saturating concentration of the MAb to LFA-1 the plasma neutralized both viruses by more than 80% at this dilution. Immunoglobulin G purified from AIDS plasma, when used in combination with the MAb to LFA-1, showed the same synergistic effect in HIV neutralization as seen with the AIDS plasma and anti-LFA-1. The MAb against LFA-1 partially neutralized both viral isolates (45 to 55%) on its own. These results demonstrate significant synergy between the plasma and antibody against LFA-1 in the neutralization of HIV. The observations therefore suggest an important role for adhesion molecules in HIV infectivity and transmission. The results have implications for the recently observed host effect on HIV susceptibility to antibody neutralization.     

Autophagy pathway intersects with HIV-1 biosynthesis and regulates viral yields in macrophages

Autophagy is a cytoplasmic degradative pathway that can participate in biosynthetic processes, as in the yeast Cvt pathway, but is more commonly known for its functions in removing damaged or surplus organelles and macromolecular complexes. Here, we find that autophagy intersects with human immunodeficiency virus (HIV) biogenesis, mirroring the above dichotomy. Early, nondegradative stages of autophagy promoted HIV yields. HIV Gag-derived proteins colocalized and interacted with the autophagy factor LC3, and autophagy promoted productive Gag processing. Nevertheless, when autophagy progressed through maturation stages, HIV was degraded. This, however, does not occur, as the HIV protein Nef acts as an antiautophagic maturation factor through interactions with the autophagy regulatory factor Beclin 1, thus protecting HIV from degradation. The dual interaction of HIV with the autophagy pathway enhances viral yields by using the early stages while inhibiting the late stages of autophagy. The role of Nef in the latter process enhances yields of infectious HIV and may be of significance for progression to clinical AIDS.     

Suppression of HIV replication using RNA interference against HIV-1 integrase

RNA interference (RNAi) has become one of the most powerful and popular approach on gene silencing in clinical research study especially in virology due to the gene-specific suppression property of small interfering RNA (siRNA). In this report, we demonstrate that expression of vector-mediated small hairpin RNA (shRNA) against human immunodeficiency virus type 1 (HIV-1) integrase (IN), one of the three important enzymes in HIV infection by controlling the integration of viral RNA to host DNA, could suppress the protein synthesis of EGFP-tagged IN in HeLa cell model efficiently. Furthermore, we show that IN shRNA can successfully reduce the HIV particles production in 293T cells at the level similar to the positive control of HIV-1 tat shRNA. These results provide the therapeutic possibility of HIV replication using RNAi against HIV-1 integrase.     

Mucosal HIV-1 transmission and prevention strategies in BLT humanized mice

Clinical trials testing microbicides and related biomedical interventions to block HIV transmissions have produced contradictory results and to date it is unclear why. Further elucidation of the molecular basis of mucosal HIV transmission and extensive pharmacokinetic and pharmacodynamic analyses are essential to implementing effective prevention strategies. Animal models are of critical importance to this effort and bone marrow-liver-thymus (BLT) humanized mice have recently emerged as a powerful small animal research platform for in vivo efficacy evaluation of mucosal and parenteral HIV-1 prevention interventions. The availability of this validated system for the pre-clinical evaluation of HIV-1 prevention approaches will accelerate the implementation of the best candidate interventions into clinical trials.     

Interleukin-1 receptor antagonist gene polymorphism and circulating levels of human immunodeficiency virus type 1 RNA in Brazilian women

Interleukin-1 receptor antagonist (IL-1ra) gene polymorphisms in 83 human immunodeficiency virus (HIV)-seropositive women were evaluated. Fourteen of the subjects (16.9%) were homozygous for IL-1ra allele 2 (IL-1RN*2). These women had a lower median level of HIV RNA than did women homozygous for allele 1 (IL-1RN*1) (P = 0.01) or heterozygous for both alleles (P = 0.04). Among 46 subjects not receiving antiretroviral treatment, HIV levels were also reduced in IL-1RN*2 homozygous individuals (P < 0.05). There was no relation between IL-1ra alleles and CD4 levels.     

Clinical symptoms associated with seroconversion for HIV-1 among misusers of intravenous drugs: comparison with homosexual seroconverters and infected and non-infected intravenous drug misusers.

OBJECTIVE--To study the clinical symptoms associated with seroconversion for HIV-1 among misusers of intravenous drugs. DESIGN--Case-control study in cohorts of drug misusers and homosexual men. SETTING--Outpatient clinic, Municipal Health Service, Amsterdam. SUBJECTS--Misusers of intravenous drugs from our prospective cohort who seroconverted for HIV. Controls were drug users positive for HIV, drug users negative for HIV, and homosexual men who had seroconverted. RESULTS--Five out of 18 (28%) drug users were admitted to hospital with bacterial pneumonia in the four to six months between their last visit at which they were HIV negative and their first visit when they were HIV positive. For comparison none of the 27 homosexual men who seroconverted for HIV, three out of 177 (2%) drug users negative for HIV, and 10 out of 112 (9%) drug users positive for HIV reported bacterial pneumonia. One out of the 18 drug users who seroconverted suffered from oesophageal candidiasis at the time of seroconversion. Other clinical symptoms did not differ between drug users who seroconverted and those who remained negative for HIV, probably due to the high background morbidity among the drug users. CONCLUSIONS--Seroconversion to HIV-1 among intravenous drug misusers is associated with bacterial pneumonia. Those drug users with previously negative test results for HIV who are admitted to hospital for bacterial pneumonia should be tested to detect primary infection with HIV-1.     

SIRT1影响Tat介导的HIV-1转录

Tat蛋白在HIV的转录复制中起重要作用.它能反式激活HIV的转录,促进HIV长末端重复序列(HIV LTR)的转录和延长.Tat蛋白是去乙酰化酶SIRT1的一种重要底物.Tat的乙酰化与非乙酰化状态在激活转录过程中受高度精密调控.如果Tat乙酰化状态在转录过程中受到干扰,随后其促使的HIV转录也将受到干扰.近来发现,组蛋白去乙酰化酶SIRT1在Tat蛋白介导的反式激活HIV转录过程中起重要的调控作用.SIRT1能对乙酰化的Tat进行去乙酰化,使其能在促使HIV转录的过程中循环利用.同时Tat与SIRT1的结合也会使核转录因子NF-κB的p65亚基处于超乙酰化状态,致使病毒基因组表达.研究SIRT1与Tat的相互关系为治疗HIV提供了新的方向.     

Studies on the allostimulatory function of dendritic cells from HCV-HIV-1 co-infected patients

There is increasing recognition of the potential morbidity and mortality associated with HIV- 1 and hepatitis C (HCV) co-infection. HIV appears to adversely affect HCV disease while the reciprocal effect of HCV on HIV remains controversial.We therefore studied the effect of co-infection on dendritic cell function versus HIV infection alone, as previous work has shown that HCV impairs dendritic cell (DC) function. HIV-1 positive individuals with HCV were matched for CD4 count, HIV-1 RNA viral load and therapy, to HIV-1 positive patients without HCV. Monocyte-derived DC were generated and mixed leukocyte reactions were performed. We assessed allostimulatory capacity with and without administration of exogenous Thl cytokines, using thymidine uptake and cell division analyses with the vital dye CFSE. We found that monocyte-derived DC from co-infected individuals showed no significant differences in allostimulatory capacity to ex vivo generated DC from HIV-1 infected individuals without HCV. Unlike the situation with HCV infection alone, this impairment was not reversed by increasing concentrations of either interleukin-2 or -12. Monocyte-derived DC from HIV- 1 and HCV co-infected individuals have a similar allostimulatory capacity to DC from matched patients with HIV-1 alone. These findings are compatible with results of prior clinical studies that found no evidence that HCV co-infection altered HIV disease progression and has implications for immunotherapeutic approaches in co-infected individuals.     

RNAi as a treatment for HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1) was the first primate virus shown to be inhibited by RNA interference (RNAi). Early studies used both synthetic and promoter expressed small interfering RNAs (siRNAs) or expressed short hairpin RNAs (shRNAs) to demonstrate that this virus was susceptible to RNAi. In addition to targeting the virus itself RNAi-mediated down-regulation of cellular targets that encode receptors required for viral entry also proved to be effective. The power of RNAi as an anti-HIV agent has propelled development of RNAi-based gene therapy approaches for the treatment of HIV infection in humans. Nevertheless, extensive in vitro experimentation has revealed potential problems of viral escape mutants and other toxicities caused by the si/shRNAs. This review covers the progress and problems in the development of RNAi for the treatment of HIV infection. Potential modalities for clinical application of RNAi in the treatment of HIV-1 infection are also described.     

Human immunodeficiency virus type 1 derivative with 7% simian immunodeficiency virus genetic content is able to establish infections in pig-tailed macaques

A human immunodeficiency virus type 1 (HIV-1) derivative (HIV(NL-DT5R)) containing sequences encoding a 7-amino-acid segment of CA and the entire vif gene from simian immunodeficiency virus (SIV) was previously shown to establish spreading infections in cultured macaque peripheral blood mononuclear cells. To assess its replicative and disease-inducing properties in vivo, HIV(NL-DT5R) was inoculated into pig-tailed macaques. HIV(NL-DT5R) generated plasma viremia in all five of the monkeys and elicited humoral responses against all of the HIV-1 structural proteins but did not cause CD4(+) T-lymphocyte depletion or clinical disease. Additional adaptation will be required to optimize infectivity in vivo.     

HIV mucosal vaccine: nasal immunization with rBCG-V3J1 induces a long term V3J1 peptide-specific neutralizing immunity in Th1- and Th2-deficient conditions.

In the vaccine strategy against HIV, bacillus Calmette-Guérin (BCG), a live attenuated strain of Mycobacterium bovis, is considered to be one of potential vectors for mucosal delivery of vaccine Ag. We analyzed the induction of the Ag-specific Ab response by nasal immunization with recombinant BCG vector-based vaccine (rBCG-V3J1) that can secrete the V3 principal neutralizing epitope of HIV. Mice were nasally immunized with rBCG-V3J1 (10 microg) three times at weekly intervals. Four weeks after the initial immunization, high titers of V3J1-specific IgG Abs were seen in serum. These high levels of HIV-specific serum IgG responses were maintained for >12 mo following nasal immunization without any booster immunization. V3J1-specific IgG-producing cells were detected in mononuclear cells isolated from spleen, nasal cavity, and salivary gland of the nasally vaccinated mice. Nasal rBCG-V3J1 also induced high levels of prolonged HIV-specific serum IgG responses in Th1 (IFN-gamma(-/-))- or Th2 (IL-4(-/-))-immunodeficient mice. Further, IgG3 was highest among V3 peptide-specific IgG subclass Ab responses in these immunodeficient mice as well as in wild-type mice. In addition, this Ag-specific serum IgG Abs induced by nasal immunization with rBCG-V3J1 possessed the ability to neutralize clinical isolate of HIV in vitro. These results suggested that the nasal rBCG-V3J1 system might be used as a therapeutic vaccine in addition to a prophylaxis vaccine for the control of AIDS.     

Population HIV-1 dynamics in vivo: applicable models and inferential tools for virological data from AIDS clinical trials

In this paper, we introduce a novel application of hierarchical nonlinear mixed-effect models to HIV dynamics. We show that a simple model with a sum of exponentials can give a good fit to the observed clinical data of HIV-1 dynamics (HIV-1 RNA copies) after initiation of potent antiviral treatments and can also be justified by a biological compartment model for the interaction between HIV and its host cells. This kind of model enjoys both biological interpretability and mathematical simplicity after reparameterization and simplification. A model simplification procedure is proposed and illustrated through examples. We interpret and justify various simplified models based on clinical data taken during different phases of viral dynamics during antiviral treatments. We suggest the hierarchical nonlinear mixed-effect model approach for parameter estimation and other statistical inferences. In the context of an AIDS clinical trial involving patients treated with a combination of potent antiviral agents, we show how the models may be used to draw biologically relevant interpretations from repeated HIV-1 RNA measurements and demonstrate the potential use of the models in clinical decision-making.