Bispecific Antibodies Targeting Different Epitopes on the HIV-1 Envelope Exhibit Broad and Potent Neutralization.

UNLABELLED: The potency and breadth of the recently isolated neutralizing human monoclonal antibodies to HIV-1 have stimulated interest in their use to prevent or to treat HIV-1 infection. Due to the antigenically diverse nature of the HIV-1 envelope (Env), no single antibody is highly active against all viral strains. While the physical combination of two broadly neutralizing antibodies (bNAbs) can improve coverage against the majority of viruses, the clinical-grade manufacturing and testing of two independent antibody products are time and resource intensive. In this study, we constructed bispecific immunoglobulins (IgGs) composed of independent antigen-binding fragments with a common Fc region. We developed four different bispecific IgG variants that included antibodies targeting four major sites of HIV-1 neutralization. We show that these bispecific IgGs display features of both antibody specificities and, in some cases, display improved coverage over the individual parental antibodies. All four bispecific IgGs neutralized 94% to 97% of antigenically diverse viruses in a panel of 206 HIV-1 strains. Among the bispecific IgGs tested, VRC07 × PG9-16 displayed the most favorable neutralization profile. It was superior in breadth to either of the individual antibodies, neutralizing 97% of viruses with a median 50% inhibitory concentration (IC50) of 0.055 µg/ml. This bispecific IgG also demonstrated in vivo pharmacokinetic parameters comparable to those of the parental bNAbs when administered to rhesus macaques. These results suggest that IgG-based bispecific antibodies are promising candidates for the prevention and treatment of HIV-1 infection in humans. IMPORTANCE: To prevent or treat HIV-1 infection, antibodies must potently neutralize nearly all strains of HIV-1. Thus, the physical combination of two or more antibodies may be needed to broaden neutralization coverage and diminish the possibility of viral resistance. A bispecific antibody that has two different antibody binding arms could potentially display neutralization characteristics better than those of any single parental antibody. Here we show that bispecific antibodies contain the binding specificities of the two parental antibodies and that a single bispecific antibody can neutralize 97% of viral strains with a high overall potency. These findings support the use of bispecific antibodies for the prevention or treatment of HIV-1 infection.

Safety, pharmacokinetics and neutralization of the broadly neutralizing HIV-1 human monoclonal antibody VRC01 in healthy adults.

VRC-HIVMAB060-00-AB (VRC01) is a broadly neutralizing HIV-1 monoclonal antibody (mAb) isolated from the B cells of an HIV-infected patient. It is directed against the HIV-1 CD4 binding site and is capable of potently neutralizing the majority of diverse HIV-1 strains. This Phase I dose-escalation study in healthy adults was conducted at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA). Primary objectives were the safety, tolerability and pharmacokinetics (PK) of VRC01 intravenous (i.v.) infusion at 5, 20 or 40 mg/kg, given either once (20 mg/kg) or twice 28 days apart (all doses), and of subcutaneous (s.c.) delivery at 5 mg/kg compared to s.c. placebo given twice, 28 days apart. Cumulatively, 28 subjects received 43 VRC01 and nine received placebo administrations. There were no serious adverse events or dose-limiting toxicities. Mean 28-day serum trough concentrations after the first infusion were 35 and 57 µg/ml for groups infused with 20 mg/kg (n = 8) and 40 mg/kg (n = 5) doses, respectively. Mean 28-day trough concentrations after the second infusion were 56 and 89 µg/ml for the same two doses. Over the 5-40 mg/kg i.v. dose range (n = 18), the clearance was 0.016 l/h and terminal half-life was 15 days. After infusion VRC01 retained expected neutralizing activity in serum, and anti-VRC01 antibody responses were not detected. The human monoclonal antibody (mAb) VRC01 was well tolerated when delivered i.v. or s.c. The mAb demonstrated expected half-life and pharmacokinetics for a human immunoglobulin G. The safety and PK results support and inform VRC01 dosing schedules for planning HIV-1 prevention efficacy studies.

Influenza virus h5 DNA vaccination is immunogenic by intramuscular and intradermal routes in humans.

Avian influenza virus causes outbreaks in domestic and wild birds around the world, and sporadic human infections have been reported. A DNA vaccine encoding hemagglutinin (HA) protein from the A/Indonesia/5/05 (H5N1) strain was initially tested in two randomized phase I clinical studies. Vaccine Research Center study 304 (VRC 304) was a double-blinded study with 45 subjects randomized to placebo, 1 mg of vaccine, or 4 mg of vaccine treatment groups (n = 15/group) by intramuscular (i.m.) Biojector injection. VRC 305 was an open-label study to evaluate route, with 44 subjects randomized to intradermal (i.d.) injections of 0.5 mg by needle/syringe or by Biojector or 1 mg delivered as two 0.5-mg Biojector injections in the same deltoid or as 0.5 mg in each deltoid (n = 11/group). Injections were administered at weeks 0, 4, and 8 in both studies. Antibody responses to H5 were assessed by hemagglutination inhibition (HAI) assay, enzyme-linked immunosorbent assay (ELISA), and neutralization assay, and the H5 T cell responses were assessed by enzyme-linked immunospot and intracellular cytokine staining assays. There were no vaccine-related serious adverse events, and the vaccine was well tolerated in all groups. At 1 mg, i.d. vaccination compared to i.m. vaccination induced a greater frequency and magnitude of response by ELISA, but there were no significant differences in the frequency or magnitude of response between the i.d. and i.m. routes in the HAI or neutralization assays. T cell responses were more common in subjects who received the 1- or 4-mg dose i.m. These studies demonstrated that the DNA vaccine encoding H5 is safe and immunogenic and served to define the proper dose and route for further studies. The i.d. injection route did not offer a significant advantage over the i.m. route, and no difference was detected by delivery to one site versus splitting the dose between two sites for i.d. vaccine administration. The 4-mg dose (i.m) was further investigated in prime-boost regimens.

A replication defective recombinant Ad5 vaccine expressing Ebola virus GP is safe and immunogenic in healthy adults.

Ebola virus causes irregular outbreaks of severe hemorrhagic fever in equatorial Africa. Case mortality remains high; there is no effective treatment and outbreaks are sporadic and unpredictable. Studies of Ebola virus vaccine platforms in non-human primates have established that the induction of protective immunity is possible and safety and human immunogenicity has been demonstrated in a previous Phase I clinical trial of a 1st generation Ebola DNA vaccine. We now report the safety and immunogenicity of a recombinant adenovirus serotype 5 (rAd5) vaccine encoding the envelope glycoprotein (GP) from the Zaire and Sudan Ebola virus species, in a randomized, placebo-controlled, double-blinded, dose escalation, Phase I human study. Thirty-one healthy adults received vaccine at 2×10(9) (n=12), or 2×10(10) (n=11) viral particles or placebo (n=8) as an intramuscular injection. Antibody responses were assessed by ELISA and neutralizing assays; and T cell responses were assessed by ELISpot and intracellular cytokine staining assays. This recombinant Ebola virus vaccine was safe and subjects developed antigen specific humoral and cellular immune responses.

CCR5 and CXCR4 expression correlated with X4 and R5 HIV-1 infection yet not sustained replication in Th1 and Th2 cells.

OBJECTIVE: To investigate the infectivity of T-helper (Th)1 and Th2 cells (derived from ccr5 wild-type and homozygous ccr5 Delta 32) to R5 and X4 HIV-1. DESIGN: It remains unclear whether infection of Th1 and Th2 CD4 cells by R5 and X4 viruses mirrors their co-receptor expression profile as no direct quantitation of coreceptor levels and infection has been performed. In addition, it is unknown whether the lack of CCR5 expression affects the degree of Th1/Th2 polarization. METHODS: Surface expression of CCR5 and CXCR4 was determined by quantitative fluorescence activated cell sorter analysis on in vitro differentiated Th1 and Th2 cells. R5 (Ba-L) and X4 (IIIB) HIV-1 isolates were used for infection studies and the efficiency of viral entry was determined by quantitative real time polymerase chain reaction detection of reverse transcribed proviral DNA. RESULTS: Cell surface density of CCR5 molecules was eight-fold higher in Th1 versus Th2 subsets (P = 0.005) whereas CXCR4 surface density was four-fold higher in Th2 versus Th1 subsets (P = 0.006). Preferential infection and entry of Th1 cells by R5 HIV-1 was not associated with preferential replication, as eventually the R5-virus replicated to a higher level in Th2 cells in spite of lower initial viral infection/entry. By contrast, Th2 cells preferentially supported X4-virus infection and replication. High beta chemokine secretion by Th1 cells was associated with a lower R5 replication rate. CONCLUSIONS: Th1 and Th2 cells differ in their infection efficiency for R5 and X4 HIV-1. ccr5 Delta 32-homozygous individuals maintain the ability for Th1/Th2 polarization, i.e., the expression of CCR5 is not required for Th1/Th2 polarization.

IL-13 and TNF-alpha inhibit dual-tropic HIV-1 in primary macrophages by reduction of surface expression of CD4, chemokine receptors CCR5, CXCR4 and post-entry viral gene expression.

We show that IL-13 in the presence of TNF-alpha effected an equal or greater antiviral activity against a dual-tropic HIV-1 (R5X4) in macrophages. A temporary or continued exposure of macrophages to both cytokines significantly decreased the infection and replication of R5X4 HIV-1(89.6) (median, 128-fold, n = 9, p = 0.024) in macrophages as compared to untreated controls when analyzed over six decreasing multiplicities of infection. A quantitative flow cytometric assay revealed that IL-13 induced a significant (approximately 50 %) reduction in the number of CD4 and CC chemokine receptor 5 (CCR5) antibody binding sites while completely abrogating surface expression of CXC chemokine receptor 4 (CXCR4). In the presence of IL-13 and TNF-alpha, expression of CCR5 was completely abrogated while the expression of CD4 and CXCR4 remained significantly reduced as compared to untreated controls. A reduction in CD4 and HIV-1 coreceptors was associated with a decrease in reverse-transcribed viral DNA at 24 h post-infection. Quantification of viral gene expression using amphotropic MLV Env pseudotyped luciferase reporter viruses suggested that IL-13 inhibited HIV-1 gene expression within 24 h by up to 90 % in the presence or absence of TNF-alpha. In conclusion, our data suggest that IL-13 is a powerful counter-regulatory agent against TNF-alpha-induced HIV-1 expression while also acting with TNF-alpha in inhibiting de novo infection of macrophages.

Inhibition of IL-12 production in human monocyte-derived macrophages by TNF.

IL-12 is a pivotal cytokine that links the innate and adaptive immune responses. TNF-alpha also plays a key role in orchestrating inflammation and immunity. The reciprocal influence of these two inflammatory mediators on each other may have significant impact on the cytokine balance that shapes the type and extent of immune responses. To investigate the relationship between TNF-alpha and IL-12 production, we analyzed the effects of exposure of human monocyte-derived macrophages to TNF-alpha on LPS- or Staphylococcus aureus-induced IL-12 production in the presence or absence of IFN-gamma. TNF-alpha is a potent inhibitor of IL-12 p40 and p70 secretion from human macrophages induced by LPS or S. aureus. IL-10 is not responsible for the TNF-alpha-mediated inhibition of IL-12. TNF-alpha selectively inhibits IL-12 p40 steady-state mRNA, but not those of IL-12 p35, IL-1alpha, IL-1beta, or IL-6. Nuclear run-on analysis identified this specific inhibitory effect at the transcriptional level for IL-12 p40 without down-regulation of the IL-12 p35 gene. The major transcriptional factors identified to be involved in the regulation of IL-12 p40 gene expression by LPS and IFN-gamma, i.e., c-Rel, NF-kappaB p50 and p65, IFN regulatory factor-1, and ets-2, were not affected by TNF-alpha when examined by nuclear translocation and DNA binding. These data demonstrate a selective negative regulation on IL-12 by TNF-alpha, identifying a direct negative feedback mechanism for inflammation-induced suppression of IL-12 gene expression.

IL-13 and IFN-gamma secretion by activated T cells in HIV-1 infection associated with viral suppression and a lack of disease progression.

The immunopathology of HIV-1 infection includes immune defects in T cell cytokine secretion, resulting in decreased Ag-specific responses. In this report, IL-13 and IFN-gamma were analyzed in progressive HIV-1 disease. Both cytokines exert positive effects on Ag presentation and inhibit HIV-1 infection of macrophages. Anti-CD3/anti-CD28-activated PBMC from HIV-1-infected individuals (n = 74) compared with uninfected subjects (n = 30) secreted significantly less IL-13 (median, 0.64 ng/ml vs 2.07 ng/ml; p < 0. 001) and IFN-gamma (median, 40.96 ng/ml vs 129.5 ng/ml; p < 0.005). Decreased IL-13 and IFN-gamma secretion in HIV infection was present in sorted CD4+ and CD8+ T cell subsets, and additional analysis determined concurrent deficiency at the protein and transcriptional level. Longitudinal analysis showed that cytokine secretion levels correlated positively with CD4 count and negatively with plasma HIV-1 viral load. Patients changing to suppressive antiretroviral therapy during the study showed increases in IL-13 and IFN-gamma secretion. Overall, results show a decline in IL-13 and IFN-gamma secretion in progressive HIV-1 infection and suggest a role for both cytokines as part of T cell adaptive responses associated with a lack of disease progression.

IL-13 acts on macrophages to block the completion of reverse transcription, inhibit virus production, and reduce virus infectivity.

An understanding of the immune suppression of HIV-1 replication in macrophages continues to be a major goal of AIDS research due to the central role this cell type has in AIDS pathogenesis. We have previously discussed the potential clinical benefits of the anti-inflammatory cytokine interleukin-13 (IL-13), which, unlike IL-4 or IL-10, had limited effects on T cell functions. In this report are extend our observations on the effects of IL-13 on HIV-1 replication in monocyte-derived macrophages (MDM) and show redundancy with IL-4, IL-13 or IL-4 have similar effects on HIV-1 replication in MDM when added at different times after infection, with the ability to decrease infection virus release when added for up to 7 days after infection. Removal of IL-13 from MDM revealed a reduction of infection by 16- to 81-fold based on the absence of viral re-emergence from lower multiplicity of infection (m.o.i.). The reduction of HIV-1 infectivity in MDM caused by IL-13 was further characterized by studies on the formation of viral DNA over a range of m.o.i. IL-13 increased the formation of LTR DNA at the lowest m.o.i. of 0.007 while concurrently inhibiting the formation of gag DNA, a later reverse transcription product, at the highest m.o.i. tested, 0.62. Overall, our data indicate that IL-13 can act on macrophages before and after HIV-1 infection by blocking the completion of reverse transcription, decreasing virus production, and reducing the infectivity of the progeny virions.

Expression of interleukin-6 receptors and NF-kappa B in AIDS-related Kaposi sarcoma cell strains.

AIDS-related Kaposi sarcoma (AIDS-KS) is the most common malignancy associated with HIV infection, with an incidence of 10-30% of all AIDS patients. As such, there have been a large number of AIDS-KS cell strains isolated and numerous studies conducted to elucidate the mechanisms of malignancy in this disease. We have reported histological grade associated differences in the ability of AIDS-KS cell strains to proliferate under conditions of minimal growth factor supplementation, with strains derived from high grade lesions having enhanced proliferation potential. Furthermore, we found that this difference in in vitro growth characteristics was not attributed to grade associated differences in autologous growth factor release. These current investigations explored the hypothesis that grade associated growth differences could be attributed to differences in the expression of the components of the IL-6 receptor, or expression/inducibility of the pleotrophic transcription factor NF-kappa B. We determined there were no significant grade associated differences in the expression of either component (IL-6R alpha chain or gpl30) of the IL-6 receptor. However, non-lesional oral derived cell strain lysates from AIDS-KS patients (n = 4) contained significantly lower concentrations of both components of the IL-6 receptor than AIDS-KS strains (n = 8) and lower concentrations of gp-130 than normal human oral derived fibroblasts (n = 2). Comparative analysis of sera concentrations of soluble components of the IL-6 receptor did not demonstrate significant differences between HIV+/KS+ (n = 7), HIV+/KS- (n = 9) and normal (HIV-/KS-) (n = 4) populations. Further, no differences were detected in the expression of NF-kappa B in AIDS-KS cell strains (n = 5) derived from both high and low histological grade lesions as compared to nonlesional AIDS-KS cell stain (n = 1) and normal human oral derived fibroblasts (n = 2) under conditions of: constitutive/proliferative growth, sera starvation, oxidative stress, and mitogen reintroduction after sera starvation. In conclusion, these investigations have eliminated two explanations for histological grade associated differences for in vitro growth potential of AIDS-related KS cell strains and further substantiated the lack of systemic paracrine cytokine/cytokine receptor effects in AIDS-KS pathogenesis.

Constitutive cytokine release by simian T-cell lymphotrophic virus type I (STLV-I) and human T-cell lymphotrophic virus types I/II (HTLV-I/II) transformed cell lines.

Simian T-cell lymphotrophic virus type I (STLV-I) and human T-cell lymphotrophic virus types I/II (HTLV-I/II) contain the tax gene which can transactivate the transcription of viral and cellular genes including several cytokines. These investigations used two STLV-I and four different HTLV-I/II transformed cell lines to quantitate constitutive cytokine release, p24 antigen production, and to correlate gag p24 antigen and cytokine release. These investigations are the first to report 1) quantitative comparison of constitutive release of multiple cytokines by several STLV-I and HTLV-I/II transformed cell lines and to determine the cytokines constitutively released by STLV-I transformed cell lines as IL-6, b-FGF, and GM-CSF (and TNF-beta, and PDGF in a higher viral producing line); 2) statistically significant differences in levels of cytokines produced by STLV-I and HTLV-I/II transformed cell lines, dependent on the method of results quantification; and 3) a correlation between levels of STLV-I and HTLV-I/II and gag p24 antigen production and cytokine production.

Cultured AIDS-related Kaposi's sarcoma (AIDS-KS) cells demonstrate impaired bioenergetic adaptation to oxidant challenge: implication for oxidant stress in AIDS-KS pathogenesis.

Despite its recognition as the most prevalent HIV associated cancer, speculation still abounds regarding the pathogenesis of AIDS-related Kaposi's sarcoma (AIDS-KS). However, it has been established that both cytokines, e.g. IL-6, and HIV-associated products, e.g., Tat, are integral in AIDS-KS cellular proliferation. Further, both experimental and clinical evidence is accumulating to link reactive oxygen intermediates (ROI) with both cytokine induction (primarily via nuclear factor-kappa B[NF-kappa B] dependent routes) as well as the subsequent cytokine, tumor necrosis factor alpha (TNF alpha) stimulation of HIV replication. Features of AIDS-KS patients, such as retention of phagocytes, presence of sustained immunostimulation, and a frequent history of KS lesions arising at traumatized sites, make oxidant stress a viable clinical factor in AIDS-KS development. Time course nucleotide profile analyses show that AIDS-KS cells have an inherent, statistically significant, biochemical deficit, even prior to oxidant stress, due to 1) a more glycolytic bioenergetic profile, resulting in lower levels of high energy phosphates (impairing capacity for glutathione [GSH] synthesis and DNA repair); 2) lower levels of NADPH (compromising the activities of GSSG reductase and peroxidase function of catalase); and 3) reduced levels of GSH (impeding both GSH peroxidase and GSH-S-transferases). Following exposure to physiologically relevant levels of H2O2, only the human microvascular endothelial cells (a putative AIDS-KS progenitor cell) responded with bioenergetic adaptations that reflected co-ordination of energy generating and cytoprotective pathways, e.g., retention of the cellular energy charge, increased NAD+, and an accentuation of the ATP, NADPH, and total adenine nucleotide differences relative to AIDS-KS cells. Also, some of the AIDS-KS strains retained intracellular GSSG subsequent to oxidant challenge, inviting the formation of deleterious protein mixed disulfides. While the results of our study address some AIDS-KS issues, they also raise an etiological question, i.e., Does the inability to tolerate oxidant stress arise in conjunction with AIDS-KS neoplastic development, or is it pre-existing in the population at risk? Regardless, use of antioxidant therapy (low risk/ potentially high benefit) in both the "at risk" population as well as in those individuals with active disease may prove a useful preventative and/or treatment modality.

Correlation between AIDS-related Kaposi sarcoma histological grade and in vitro behavior: reduced exogenous growth factor requirements for isolates from high grade lesions.

Kaposi sarcoma, the most common AIDS-associated malignancy, affects 10-30% of all AIDS patients. To date, research into the biological characteristics of AIDS-related Kaposi sarcoma (AIDS-KS) derived cell lines has been based on cultures established from skin explants or pleural effusions/peritoneal fluids. We have established several AIDS-KS lines from biopsy confirmed oral mucosal and epidermal AIDS-KS lesions and have found a correlation between AIDS-KS lesional grade and in vitro cellular growth characteristics. In comparison to epidermal AIDS-KS lesions, mucosal AIDS-KS lesions frequently possessed both a more advanced histologic grade and demonstrated a greater capacity to proliferate in minimal medium. We report the ability of AIDS-KS isolates from high grade lesions to sustain proliferation (greater than 60 population doubling levels) in medium not supplemented with endothelial cell growth supplement and/or cytokine rich conditioned medium. These findings indicate that AIDS-KS cells isolated from high grade lesions have reduced requirements for exogenously provided growth supplements, and suggest that increased autologous cytokine production accompanies AIDS-KS lesional progression.

Comparison of constitutive cytokine release in high and low histologic grade AIDS-related Kaposi's sarcoma cell strains and in sera from HIV+/KS+ and HIV+/KS- patients.

Kaposi's sarcoma (KS) is both an AIDS-defining disease and the most common HIV-associated malignancy. A cytokine-mediated pathogenesis for AIDS-KS is implicated because AIDS-KS-derived cell strains both respond to and express a variety of cytokines. We have reported the establishment of several (n = 18) AIDS-KS cell strains and determined that reduced exogenous growth factors are necessary to sustain proliferation in isolates from high histologic grade KS lesions. This current investigation explored the possibility that there are histologic grade-associated differences in either the qualitative and/or quantitative constitutive release of AIDS-KS growth stimulatory cytokines. Our findings showed that the incorporation of HTLV-II cytokine-rich conditioned media induced both qualitative and significant quantitative cytokine release, suggesting that exogenous growth promoters stimulate constitutive cytokine release. ELISA of our AIDS-KS cell strains demonstrated constitutive release of IL-6 (seven of seven), FGF-2 (five of seven), GM-CSF (three of seven), and IL-1 beta (one of seven). None of our AIDS-KS cell strains constitutively released detectable levels of Onco-M, IL-4, PDGF, TNF-alpha, or TNF-beta. In addition, we report that the method of cytokine result quantitation significantly affects reported cytokine levels. We determined that there was no significant histologic grade-dependent difference in the constitutive release of soluble cytokines by in vitro grown cultures of AIDS-KS cells. The presence of HIV influenced the sera cytokine profiles by elevating IL-6 and decreasing PDGF concentrations of HIV+ individuals relative to HIV- healthy controls. However, the presence of KS was not associated with unique serum cytokine profiles, because no differences were noted in comparisons of HIV+/KS+ versus HIV+/KS- individuals. Our findings suggest that the local environment is key in modulating AIDS-KS cytokine expression and that KS growth-promoting factors function at the local or paracrine, not the systemic, level. In conclusion, our previous results demonstrated a histologic grade-associated difference in the in vitro growth capacity of AIDS-KS cells; with high histologic grade isolates displaying a marked growth advantage during culture in minimally supplemented media. Findings from this current study reveal that although the potential for a constitutive growth loop exists in the high-grade isolates, it is not reflected in the free levels of soluble cytokines secreted into the culture medium.

Immune recognition of genetically diverse simian T-cell lymphotropic virus type I isolates.

Nucleotide sequence analysis of selected regions of the gag, pol, env and pX genes of simian T-cell lymphotropic virus type I (STLV-I) strains indicated that African isolates were more closely related to human T-cell lymphotropic virus type I (HTLV-I) than Asian isolates. Despite these recent comparative studies on nucleotide sequence homology between HTLV-I and STLV-I isolates, only limited information is available regarding the influence of genetic differences on antigen-antibody recognition of distinct STLV-I strains. In this study, we demonstrated that sera from STLV-I-infected yellow baboons (Papio cynocephalus) reacted strongly with env gp62/68 from HTLV-I-infected cell lines MT-2 and C10/MJ. In contrast, sera from Japanese macaques (Macaca fuscata) naturally infected with Asian STLV-I had weak reactivity to env gp62/68 of these prototypic HTLV-I strains. Pst-1 restriction enzyme analysis of proviral DNA indicated that the baboon virus isolates were more closely related to HTLV-I than the Japanese isolates. These results indicate that nucleotide sequence diversity, correlates with variations in proviral restriction enzyme sites and antibody recognition of viral envelope proteins. These differences in immunoreactivity may have important implications for serologic diagnosis, as well as epidemiological and vaccine studies of STLV-I infection.

Lack of evidence for human T cell lymphotrophic virus type I or II infection in patients with systemic lupus erythematosus or rheumatoid arthritis.

OBJECTIVE: Human retroviruses including human immunodeficiency virus (HIV) and human T cell lymphotrophic virus Types I and II (HTLV-I/II) have been associated with forms of connective tissue autoimmune diseases including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). We looked for evidence of HTLV-I/II infection in a large population of SLE, RA, and control patients. METHODS: One hundred fifteen patients with connective tissue autoimmune disease and other rheumatological disorders were screened for antibodies to HTLV-I/II by Western immunoblots (WIB). Due to the transforming characteristic of these retroviruses, the patients' peripheral blood mononuclear cells (PBMNC) were cultured in attempts to establish continuous cell lines. Furthermore, PBMNC culture supernatants were analyzed for reverse transcriptase activity and/or HTLV-I/II gag antigen production. The presence of HTLV-I/II proviral sequences in short term culture and fresh PBMNC was determined by Southern blot analysis and polymerase chain reaction (PCR), respectively. respectively. RESULTS: All 115 patients were HTLV-I/II and HIV seronegative. Seventy-four attempts to establish PBMNC cell lines from 65 patients were unsuccessful with a mean culture survival time of 3.6 (+/- 1.4) months. Reverse transcriptase activity and HTLV-I/II gag antigen production were not detected in 51 and 16 culture supernatants tested, respectively. Cells from 11 patients tested by Southern blot analysis and from 57 patients tested by PCR were negative for HTLV-I/II related sequences. CONCLUSION: Our results failed to establish an association between human retroviruses (HTLV-I/II and HIV) and SLE, RA, or other rheumatological disorders. However, these results do not rule out other exogenous or endogenous retroviruses that may play a role in the initiation and/or promotion of these diseases.

Cultured AIDS-related Kaposi's sarcoma cells retain a proliferative bioenergetic profile but demonstrate reduced cytoprotective capabilities.

Features of AIDS-related Kaposi's sarcoma (AIDS-KS), such as the multifocal presentation at mucosal and epidermal sites subjected to trauma, suggest that AIDS-KS is initially a reactive hyperplasia that subsequently progresses to a neoplasia. It is recognized that there is an association between sustained inflammatory states and the subsequent development of neoplasia (e.g., ulcerative colitis/colonic adenocarcinoma). Furthermore, patients who develop AIDS-KS experience both a constant immune stimulation due to sustained high levels of virus-induced cytokines and, because of a sparing effect on their phagocytic cells, retention of the phagocytic inflammatory response. A component of phagocytic activation is the initiation of the oxidative burst, resulting in the generation of reactive oxygen species (ROS), which can be mutagenic to host cells if released beyond the phagolysosome and not inactivated. Our results demonstrate that cultured AIDS-KS cells possess decreased cytoprotective capabilities. Relative to either dermal fibroblasts, or human microvascular endothelial cells (HMECs), AIDS-KS cells contained significantly lower levels of glutathione, a tripeptide integral in both cytoprotection and maintenance of cellular thiol status. While HMECs increased catalase activity during culture in the cytokine-rich KS milieu (control medium supplemented with conditioned medium from MOT, an HTLV II-infected cell line), AIDS-KS cells demonstrated reduced catalase function under these conditions. Furthermore, HMEC cultures showed an inherent biochemical responsiveness, by increasing catalase activity following exposure to exogenous H2O2. In contrast, the catalase activity of AIDS-KS cells decreased following H2O2 challenge. Our results show that an inherent deficiency in cellular cytoprotection is present in AIDS-KS cells and suggest that oxidant stress may function in the development and progression of AIDS-KS.

Sexual transmission of simian T-lymphotropic virus type I: a model of human T-lymphotropic virus type I infection.

Simian T-lymphotropic virus type-I (STLV-I) seronegative females placed together with seropositive males for breeding purposes were followed from 1984-1990 to determined seroconversion rates by enzyme immunoassay and western immunoblot analysis. Two of 26 females and 1 of 4 males previously negative for antibodies to STLV-I seroconverted during the study period. Statistical analysis of sexual encounters indicated that the probability of a seronegative female testing positive for STLV-I after a sexual encounter with a seropositive male is less than 4%. These data indicate that even though sexual contact is important in the transmission of STLV-I, it may not be an efficient mode of viral infection. These data also suggest that female-to-male transmission of STLV-I occurs, as recently reported for human T-lymphotropic virus type-I (HTLV-I) infection. These results are important because HTLV-I and STLV-I share many features in common including routes of viral transmission. In addition, the difficulty of clearly quantitating the risk of sexual transmission in humans makes the primate animal model a valuable alternative to study the human infection.

Fusion activity dissociated from replication ability in feline immunodeficiency virus (FIV) in human cells.

Multinucleated-giant-cell formation followed by cell killing was observed after cocultivation of the feline immunodeficiency virus (FIV)-producing feline T-cell line 3201/FIV with various human cells, including T-cell lines carrying human T-cell lymphotropic virus type I (HTLV-I). The susceptibility to giant cell formation varied with the cell lines tested. Cocultivation of irradiated 3201/FIV cells with MT-2 cells resulted in giant cell formation as early as 2 h in culture, with striking resemblance to that induced by human immunodeficiency virus (HIV). MT-4 cells (HTLV-I positive) and H9 cells (HTLV-I negative) were less susceptible than MT-2 to the induction of syncytia. MOLT-4 cells (HTLV-I negative) had intermediate sensitivity to syncytia formation. No syncytia were observed in the monocytic cell line U-937 (HTLV-I negative). Syncytia formation between 3201/FIV and MT-2 cells was inhibited by polyclonal cat anti-FIV antisera but not polyclonal cat anti-feline leukemia virus (FeLV) antisera, goat anti-FeLV, uninfected specific-pathogen-free cat serum, human anti-HTLV-I antisera, or normal human and goat serum. Concentrated cell-free FIV supernatant from 3201/FIV also induced giant cells of MT-2 cells that were indistinguishable from those induced by cocultivation. Giant cells and extensive cell killing associated with giant cell formation declined and disappeared within 10 days. Surviving cells appeared to be of normal size and grew continuously without expressing FIV antigen or releasing infective virus. Although Southern blot analysis using probes specific for FIV could not detect proviral DNA in any of the five human cell lines cocultured with irradiated 3201/FIV cells, the polymerase chain reaction (PCR) technique detected FIV-specific DNA in MOLT-4 cells. DNA from the FIV-PCR positive MOLT-4 cells was PCR negative for endogenous FeLV-specific sequences, indicating that the MOLT-4 cell DNA was not contaminated with DNA from feline cells (i.e., 3201 cells). The FIV-MOLT-4 cells remained PCR positive for FIV after 40 passages, suggesting stable integration in the human cell line. These findings indicate that FIV is capable of forming proviral DNA in human T-lymphoid cells by cocultivation, although this FIV-carrying human cell line failed to produce replication-competent viruses.

Susceptibility testing of macrolide antibiotics against Haemophilus influenzae and correlation of in vitro results with in vivo efficacy in a mouse septicemia model.

There is poor correlation between the MICs and zone sizes obtained for erythromycin against Haemophilus influenzae. The effect of two media, Mueller-Hinton medium supplemented with 3% lysed horse blood and 10 micrograms of NAD per ml (MHA + LYHB) and Mueller-Hinton agar supplemented with 1% bovine hemoglobin and 1% IsoVitaleX (MHA + HGB), on the MICs and zone sizes of erythromycin against H. influenzae was determined. The effect of three different methods for inoculum preparation on the susceptibility of H. influenzae was also determined. The MICs were independent of the method of inoculum preparation, but the zone sizes were smaller if the inoculum was carefully adjusted to contain approximately 10(8) CFU/ml. MICs were higher and zone sizes were smaller when MHA + HGB was used instead of MHA + LYHB. Good correlation was found when MHA + LYHB was used for determining the MIC and MHA + HGB was used for determining susceptibility by the disk method. When the inoculum was adjusted to match a McFarland 0.5 standard, the viable counts had to be approximately 10(8) CFU/ml for good correlation between MICs and zone sizes. A-56268, a new macrolide antibiotic, was tested against H. influenzae, and its MICs and tentative breakpoints against this organism were determined. The MICs obtained by various methods were correlated with in vivo efficacy by using a mouse septicemia model. MICs obtained on MHA + HGB or MHA + LYHB incubated without a 5% CO2 atmosphere showed the best correlation with in vivo efficacy.