Cystatin B and HIV regulate the STAT-1 signaling circuit in HIV-infected and INF-ß-treated human macrophages.

Cystatin B is a cysteine protease inhibitor that induces HIV replication in monocyte-derived macrophages (MDM). This protein interacts with signal transducer and activator of transcription (STAT-1) factor and inhibits the interferon (IFN-ß) response in Vero cells by preventing STAT-1 translocation to the nucleus. Cystatin B also decreases the levels of tyrosine-phosphorylated STAT-1 (STAT-1PY). However, the mechanisms of cystatin B regulation on STAT-1 phosphorylation in MDM are unknown. We hypothesized that cystatin B inhibits IFN-ß antiviral responses and induces HIV replication in macrophage reservoirs through the inhibition of STAT-1 phosphorylation. Macrophages were transfected with cystatin B siRNA prior to interferon-ß treatment or infected with HIV-ADA to determine the effect of cystatin B modulation in STAT-1 localization and activation using immunofluorescence and proximity ligation assays. Cystatin B decreased STAT-1PY and its transportation to the nucleus, while HIV infection retained unphosphorylated STAT (USTAT-1) in the nucleus avoiding its exit to the cytoplasm for eventual phosphorylation. In IFN-ß-treated MDM, cystatin B inhibited the nuclear translocation of both, USTAT-1 and STAT-1PY. These results demonstrate that cystatin B interferes with the STAT-1 signaling and IFN-ß-antiviral responses perpetuating HIV in macrophage reservoirs.

HIV-1-negative female sex workers sustain high cervical IFNɛ, low immune activation, and low expression of HIV-1-required host genes.

Sex workers practicing in high HIV endemic areas have been extensively targeted to test anti-HIV prophylactic strategies. We hypothesize that in women with high levels of genital exposure to semen changes in cervico-vaginal mucosal and/or systemic immune activation will contribute to a decreased susceptibility to HIV-1 infection. To address this question, we assessed sexual activity and immune activation status (in peripheral blood), as well as cellular infiltrates and gene expression in ectocervical mucosa biopsies in female sex workers (FSWs; n=50), as compared with control women (CG; n=32). FSWs had low-to-absent HIV-1-specific immune responses with significantly lower CD38 expression on circulating CD4(+) or CD8(+) T-cells (both: P<0.001) together with lower cervical gene expression of genes associated with leukocyte homing and chemotaxis. FSWs also had increased levels of interferon-ɛ (IFNɛ) gene and protein expression in the cervical epithelium together with reduced expression of genes associated with HIV-1 integration and replication. A correlative relationship between semen exposure and elevated type-1 IFN expression in FSWs was also established. Overall, our data suggest that long-term condomless sex work can result in multiple changes within the cervico-vaginal compartment that would contribute to sustaining a lower susceptibility for HIV-1 infection in the absence of HIV-specific responses.

Spanish validation of the HIV dementia scale in women.

HIV infection is increasing in minority groups, particularly in African American and Hispanic women. Although the incidence of HIV dementia has decreased since the advent of highly active antiretroviral treatment, prevalence of neurocognitive complications has increased as patients are now living longer. This study's purpose was to determine the psychometric properties of the Spanish-language HIV Dementia Scale (HDS) in a group of HIV-infected women. We recruited 96 women: 60 HIV-seropositive and 36 HIV-seronegative. Modification of the HDS into a Spanish-language version consisted of translating the instructions, substituting four words in Spanish (gato, media, azul, piña), increasing 1 second in the psychomotor speed because the Spanish alphabet has more letters than the English alphabet, and not offering clues for memory recall. Cognitive impairment (CI) was defined according to the modified American Academy of Neurology HIV-dementia criteria including an asymptomatic CI group. Statistical analysis consisted of analysis of variance to determine group differences and receiver operator characteristics (ROC) to determine the optimal cutoff point for the screening of CI. HDS-Spanish total score and subscores for psychomotor speed and memory recall showed significant differences between HIV-seronegative and women with HIV-dementia (p < 0.001) and between HIV-seropositive women with normal cognition and those with HIV-dementia (p < 0.001). The optimal cutoff point of 13 or less had performance characteristics of 87% sensitivity and 46% specificity for HIV-associated CI (50.0% positive predictive value, 85.0% negative predictive value). The HDS-Spanish translation offers a useful screening tool with value for the identification of Hispanic women at risk of developing HIV-associated symptomatic neurocognitive disturbances.

Differential distribution of antibodies to different viruses in young animals in the free-ranging rhesus macaques of Cayo Santiago.

BACKGROUND: The breeding colony of free-ranging rhesus macaques was established in 1938 in Cayo Santiago (CS) with animals collected in northern India. The seroprevalence to cercopithecine herpesvirus type 1 (B virus) and simian retroviruses has been studied previously. RESULTS: This is the first report on the seropositivity to different viruses using samples collected shortly after removing animals (n = 245) from CS. All samples were negative for measles, simian immunodeficiency virus and simian type D retroviruses. The overall prevalence of antibodies was around 50% for simian T-lymphotropic virus I (STLV-I). For B virus, the prevalence was 38%. CONCLUSIONS: Data obtained showed marked differences in the antibody distribution to B virus and STLV-I within the free-ranging colony of rhesus macaques. Implication of these data for the Specific Pathogen Free program at the Caribbean Primate Research Center are also discussed.

Protection by SIV VLP DNA prime/protein boost following mucosal SIV challenge is markedly enhanced by IL-12/GM-CSF co-administration.

The ever increasing number of people infected by human immunodeficiency virus (HIV) throughout the world renders the development of effective vaccines an urgent priority. Herein, we report on an attempt to induce and enhance antiviral responses using a deoxyribonucleic acid (DNA) prime/virus-like particles (VLP) protein boost strategy adjuvanted with interleukin (IL)-12/GM-CSF in rhesus macaques challenged with simian immunodeficiency virus (SIV). Thus, groups of monkeys were administered three consecutive doses of pVecB7 a plasmid expressing VLP with or without plasmids expressing IL-12 and GM-CSF at weeks 0, 13 and 26. The VLP boost was administered at week 39 with or without IL-12. All monkeys were challenged intrarectally with SIVsmE660 2 months following the protein boost. All except one immunized monkey became infected. While all immunized monkeys showed a marked reduction of acute viral peaks, reduction of viral load set points was only achieved in groups whose prime-boost immunizations were supplemented with IL-12/GM-CSF (prime) and/or with IL-12 (boost). Control of viremia correlated with lack of disease progression and survival. Detection of virus in rectal washes at 1 year post-challenge was only successful in monkeys whose immunizations did not include cytokine adjuvant, but these loads did not correlate with plasma viral loads. In summary, use of IL-12 and/or GM-CSF was shown to provide significant differences in the outcome of SIV challenge of prime/boost immunized monkeys.

Clone B7 cells have a single copy of SIVsmB7 integrated in chromosome 20.

B7 is the designation of a cell clone derived from the human cell line CEMx174, which was infected with SIVsmH3 clone. B7 cells chronically produce high quantities of non-infectious virus-like particles (VLP) denominated SIVsmB7. Here we report the molecular characterization of the B7 cell line. We found that B7 cells have a single copy of the SIVsmB7 provirus integrated in a noncoding region of chromosome 20 (nt 24,957 of clone RP5-963K23 on human chromosome 20q 13.11-13.2). Similarly to HIV and SIVmac, we show that integration of SIVsm results in a characteristic five base pair sequence repeat of host DNA that flanks the proviral DNA genome. Since the SIVsmB7 genome has a deletion in the IN coding sequence, the generation of this defective proviral genome most likely occurred during a faulty process of reverse transcription. Thus, these studies reveal the molecular clonality of the SIVsmB7 VLP produced by B7 cells. These genetically homogeneous VLP are useful reagents for vaccine development. In addition, these particles have been used by others (Montelaro et al.) to study the maturation of immune system responses to SIV infection.

Vaccine evaluation studies of replication-defective SIVsmB7.

Non-infectious virus-like particles of SIVsmB7 that expresses env and gag gene products but are defective in pol and vpx/vpr were assessed for their ability to induce protective immunity against infection with pathogenic SIVsmE660 in rhesus macaques. Animals were immunized in three groups: group A was primed with cell-associated SIVsmB7 and boosted with cell-free SIVsmB7; group B was primed with cell-free SIVsmB7 and boosted with cell-free SIVsmB7 conjugated to iron oxide microbeads; group C was primed with cell-free SIVsmB7 mixed with Titer Max adjuvant and boosted with cell-free SIVsmB7 mixed with SAF-M adjuvant followed by secondary boosting with cell-free SIVsmB7 conjugated to microbeads. Animals were challenged intravenously with 20 animal infectious doses of SIVsmE660 grown in rhesus peripheral blood mononuclear cells 3 weeks after final boosting. All animals became infected as evidenced by quantitative virus cultivation. Sera from immunized animals contained low-titer antibodies by ELISA and low or undetectable neutralizing antibodies on the day of challenge but strong anamnestic antibody responses were observed following challenge. Interestingly, 2 of 3 animals in group A showed evidence of transient viremia and more stable CD4 counts following challenge as compared to the other immunized animals and to non-immunized controls. Thus, immunization with cell-associated SIVsmB7 did not provide sterilizing immunity against challenge with a highly pathogenic SIV strain but might have caused virus clearance later in infection.

Properties of virus-like particles produced by SIV-chronically infected human cell clones.

SIVsm chronically infected cultures were obtained after infection of CEMX174 cells with either SIVsmH3 or SIVsmE660. These phenotypically CD4 cells, formed syncytia but only when cocultivated with CD4+ cells. Single cell clones were derived from these cultures and examined for the production of virus-specific proteins. The majority of the clones expressed SIV p27 antigen and low levels of virus reverse transcriptase activity. Western blot analysis, performed with either monoclonal or polyclonal sera, showed that a chronically infected clone (B7) produced particles which contained envelope (gp135 and gp43), gag precursors and gag proteins (p27, p16 and p8). However, these particles (SIVsmB7) lacked detectable levels of vpx and of integrase, and contained several fusion proteins which expressed viral protease antigens. This defective virus failed to infect established CD4+ cell lines, as well as primary cultures of macrophages and of peripheral blood lymphocytes, obtained both from humans and from rhesus macaques. Lack of infection correlated with lack of viral DNA detection by PCR amplification of genomic DNA extracted from these cell cultures. In addition, SIVsmB7 virus lacked infectivity in vivo. Rhesus macaques inoculated with high concentrations of SIVsmB7 showed no viremia and their PBMC were PCR negative. Thus, B7 cells produced stable, non-infectious virus mutants, which contained env and gag proteins, but lacked detectable amounts of vpx and of enzymes required for virus replication. Due to the high constitutive expression of this virus-like particle, we are now testing this preparation as a vaccine.

[Brief history of AIDS in non-human primates and its contribution to the study of acquired immunodeficiency syndrome]. / Breve historia del SIDA en los primates no humanos y su contribución al estudio del síndrome de inmunodeficiencia adquirida.

Infection of the rhesus monkey (Macaca mulatta) with retroviruses originating from African non human primates (SIV) induces in this species an acquired immunodeficiency syndrome (SAIDS) closely resembling AIDS in humans. Analogies between the SIV-rhesus system and AIDS in humans are described in this work, analyzing the close relationship existing between the HIV and SIV viruses, and the similarities between SIV disease in the rhesus and HIV disease in humans. A review of current advances in AIDS vaccine research, using the SIV-rhesus model, is also included.

A new procedure for the isolation of anti-HIV compounds (polysaccharides and polyphenols) from the marine alga Fucus vesiculosus.

Anti-HIV-active polysaccharides and polyphenols were isolated from the brown seaweed Fucus vesiculosus by hot H2O extraction of both the intact and the homogenized algae. This was followed by XAD2 chromatography and by sequential precipitation of the non-adsorbed compounds with glacial HOAc and thereafter with EtOH. The precipitate was solubilized, dialyzed against distilled H2O, and chromatographed on SP-Sephadex C25 and on QAE-Sephadex A25. This was followed by gel filtration on Sephadex G50 and Sephadex G100 and finally by hplc on a Shodex Ionpak S-804 column. For comparison, the commercial product fucoidan, a sulfated algal polysaccharide, was also further purified by the chromatographic techniques mentioned above. The isolated freeze-dried fractions obtained by these procedures were tested for inhibition of both HIV-induced syncytium formation and HIV reverse transcriptase enzyme activity. Some of these fractions inhibited both of these activities at concentrations that were not cytotoxic.

The combined assessment of cellular apoptosis, mitochondrial function and proliferative response to pokeweed mitogen has prognostic value in SIV infection.

Using the assessment of the mitochondrial metabolic activity of freshly isolated blood mononuclear cells, the flow cytometric detection of apoptosis and of the proliferative responses to PWM, SIV-infected macaques were classified in: stage 0, which included all animals with unaffected parameters, and stages 1, 2, and 3, which included animals having one, two, or all three parameters affected, respectively. This novel three-parametric staging system (ISS) provides a new prognostic tool in the longitudinal study of SIV infection.

Production of monoclonal antibodies to human immunodeficiency virus type-2.

The ROD strain of the human immunodeficiency virus type 2 (HIV-2) was used to produce monoclonal antibodies. Virus grown in CEM cells was partially purified by ultracentrifugation and solubilized in a buffer containing Triton X-100. BALB/c mice were inoculated intraperitoneally with 50 micrograms of solubilized virus preparations mixed 1:1 with complete Freund's adjuvant. Animals were boosted on day 28 and sacrificed on day 31. Spleen cells from the immunized animals were fused with SP20/Ag 14 myeloma cells and cultured in HAT medium. Following selection of the hybrids of interest by an HIV-2 ELISA procedure, hybridomas were cloned twice by limiting dilution. Six clones were found to produce antibodies that reacted with HIV-2 antigens as judged by ELISA. These antibodies were concentrated by ammonium sulfate precipitation, and analyzed by the Western blot procedure. Monoclonal antibodies specifically reactive to an HIV protein of 68 KD were obtained. These antibodies did not react with an HIV-2 band of 55 KD. These data showed that the monoclonal antibodies recognized the carboxy terminal region (the RNAse H domain) of the HIV-2 retrotranscriptase enzyme.

Preparation of an attenuated dengue 4 (341750 Carib) virus vaccine. I. Pre-clinical studies.

Dengue 4 (DEN-4) virus strain 341750 Carib was modified by serial passage in primary canine kidney (PCK) cell cultures. By the 15th PCK passage, this virus was less infectious for monkeys and resulted in a significantly reduced viremia as compared to the parent DEN-4 virus. The 30th PCK passage of DEN-4 341750 Carib was non-infectious for monkeys. A vaccine prepared at the 20th PCK passage in DBS-FRhL-2 cells stimulated the production of both neutralizing and hemagglutination inhibition antibodies in monkeys; these animals were also protected against challenge with the homologous strain as well as a heterologous strain of DEN-4. An ID50 titration in monkeys resulted in a titer of greater than 10(4) plaque-forming units (PFU) for the vaccine virus and 0.5 PFU for the parent virus. Reduced monkey infectivity of this magnitude has been correlated with human attenuation in previous dengue vaccine candidates. The DEN-4 strain 341750 Carib PCK-20/FRhL-4 vaccine has been characterized and sufficiently tested to be considered for safety and immunogenicity trials in humans.

In vitro HIV infection of primate lymphocytes.

Peripheral blood mononuclear cells (PBMC) obtained from four different primate species were tested for their respective ability to support the "in vitro" replication of the human immunodeficiency viruses, HIV-1, and HIV-2. PBMC of Cebus apella, patas (Erythrocebus patas), green (cercopithecus aethiops sabaeus) and rhesus monkeys (Macaca mulatta) were infected "in vitro" with either HIV-1 or HIV-2. Cultures were assayed weekly for particle-associated reverse transcriptase activity. Both viruses were found to be cytolytic for all these monkey's PBMC. Low levels of HIV-1 and HIV-2 infection were observed in Cebus cells. However, productive infection was only detected in HIV-2 infected rhesus PBMC. The capacity of HIV-2 to replicate in rhesus cells may provide a useful model for evaluating antiviral drugs and vaccines.

Effect of penicillin and spectinomycin given for urethritis and cervicitis with Neisseria gonorrhoeae: high prevalence of penicillin-resistant isolates.

Efficacy of single-dose spectinomycin (TRO: 2 g intramuscularly) was compared with that of aqueous procaine penicillin G (APPG:4.8 x 10(6) units) plus 1 g of probenecid for treatment of gonococcal urethritis and cervicitis. Cure rates of the 210 patients who received TRO and 190 patients who received APPG were 97.6% and 91.1%, respectively. MICs of antibiotics were determined using the agar dilution method. Those isolates with MICs of APPG of less than 1.0 microgram/ml had low failure rates (2.9%), while strains with increased resistance to APPG (MICs greater than or equal to 1.0 microgram/ml) had higher failure rates (24%). Treatment failures seen with TRO were not correlated to isolates with the higher MICs. Clinical results suggest TRO could be given for treatment of genital gonorrhoea in Puerto Rico due to the high prevalence of both chromosomally-mediated penicillin-resistant Neisseria gonorrhoeae (20%) and penicillinase-producing Neisseria gonorrhoeae (7.5%) strains and the high rate of failure seen with the use of APPG.

Interactions of HIV and STDs in a group of female prostitutes.

PIP: Incidence of HIV and of several other sexually transmitted diseases (STDs) was determined in 171 female prostitutes from 3 sites in San Juan, Puerto Rico. The sites were selected by high incidence of penicillinase-producing N. gonorrhoeae in clients of prostitutes. These women came from about a dozen different countries, mostly Latin American. 14% reported they always used condoms. Specimens were taken of blood, endocervix, cervix, rectum and oropharynx, and tested for HIV, gonorrhea, syphilis, herpes, chlamydia, hepatitis B and cytomegalovirus. 18% harbored gonorrhea, of which 13% were penicillinase positive. Syphilis occurred in 8%. Chlamydia was the most prevalent infection, in 47% of subjects. Serological evidence of hepatitis B was apparent in 53%, and of cytomegalovirus in 99%. HIV status was tested after unlinking identifying information from 80 serum samples, and 16% were confirmed HIV positive. Women from the site frequented by more street walkers than bar girls had a higher incidence of hepatitis B, and were known to be more frequent users of IV drugs. These data confirm observations made elsewhere that HIV infection may coexist with other STDs.^ieng

Dengue virus vaccine studies in Puerto Rico: a review.

This review summarizes part of the work performed at the Virology Laboratories (Department of Microbiology, University of Puerto Rico School of Medicine) with live attenuated dengue virus vaccines obtained from the Walter Reed Army Institute of Research. Vaccines were tested for their respective immunogenicity and attenuation in rhesus monkeys (Maccaca mulatta) and in mosquitoes (Toxorynchites amboinensis), respectively. This experimental model revealed that out of 6 vaccines tested, only two (DEN-2/S-1 and DEN-4 strain 341750) should be further evaluated for safety and immunogenicity. Further studies are required to develop an effective vaccine to prevent dengue fever and its hemorrhagic manifestations.