Search

BACKGROUND: The 2013–2016 Ebola virus outbreak in West Africa was the most widespread in history. In response, alive attenuated recombinant vesicular stomatitis virus (rVSV) vaccine expressing Zaire Ebolavirus glycoprotein (rVSVΔG-ZEBOV-GP) was evaluated in humans. METHODS: In a phase 1, randomized, dose-ranging, observer-blind, placebo-controlled trial, healthy adults aged 18–65 years were randomized into 4 groups of 10 to receive one of 3 vaccine doses or placebo. Follow-up visits spanned 180 days postvaccination for safety monitoring, immunogenicity testing and any rVSV virus shedding. RESULTS: Forty participants were injected with rVSVΔG-ZEBOV-GP vaccine (n = 30) or saline placebo (n = 10). No serious adverse events related to the vaccine or participant withdrawals were reported. Solicited adverse events during the 14-day follow-up period were mild to moderate and self-limited, with the exception of injection-site pain and headache. Viremia following vaccination was transient and no longer detectable after study day 3, with no virus shedding in saliva or urine. All vaccinated participants developed serum immunoglobulin G (IgG), as measured by Ebola virus envelope glycoprotein-based enzyme-linked immunosorbent assay (ELISA). Immunogenicity was comparable across all dose groups, and sustained IgG titers were detectable through to the last visit, at study day 180. INTERPRETATION: In this phase 1 study, there were no safety concerns after a single dose of rVSVΔG-ZEBOV-GP vaccine. IgG ELISA showed persistent high titers at 180 days postimmunization. There was a period of reactogenicity, but in general, the vaccine was well tolerated. This study provides evidence of the safety and immunogenicity of rVSVΔG-ZEBOV-GP vaccine and importance of its further investigation. Trial registration: Clinical-Trials.gov no., NCT02374385

On August 22–23, 2013, agencies within the United States Department of Defense (DoD) and the Department of Health and Human Services (HHS) sponsored the Filovirus Medical Countermeasures (MCMs) Workshop as an extension of the activities of the Filovirus Animal Non-clinical Group (FANG). The FANG is a federally-recognized multi-Agency group established in 2011 to coordinate and facilitate U.S. government (USG) efforts to develop filovirus MCMs. The workshop brought together government, academic and industry experts to consider the needs for filovirus MCMs and evaluate the status of the product development pipeline. This report summarizes speaker presentations and highlights progress and challenges remaining in the field.

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection in infants, immunocompromised patients, and the elderly. The RSV fusion (F) protein mediates fusion of the viral envelope with the target cell membrane during virus entry and is a primary target for antiviral drug and vaccine development. The F protein contains two heptad repeat regions, HR1 and HR2. Peptides corresponding to these regions form a six-helix bundle structure that is thought to play a critical role in membrane fusion. However, characterization of six-helix bundle formation in native RSV F protein has been hindered by the fact that a trigger for F protein conformational change has yet to be identified. Here we demonstrate that RSV F protein on the surface of infected cells undergoes a conformational change following exposure to elevated temperature, resulting in the formation of the six-helix bundle structure. We first generated and characterized six-helix bundle-specific antibodies raised against recombinant peptides modeling the RSV F protein six-helix bundle structure. We then used these antibodies as probes to monitor RSV F protein six-helix bundle formation in response to a diverse array of potential triggers of conformational changes. We found that exposure of ‘membrane-anchored’ RSV F protein to elevated temperature (45–55 °C) was sufficient to trigger six-helix bundle formation. Antibody binding to the six-helix bundle conformation was detected by both flow cytometry and cell-surface immunoprecipitation of the RSV F protein. None of the other treatments, including interaction with a number of potential receptors, resulted in significant binding by six-helix bundle-specific antibodies. We conclude that native, untriggered RSV F protein exists in a metastable state that can be converted in vitro to the more stable, fusogenic six-helix bundle conformation by an increase in thermal energy. These findings help to better define the mechanism of RSV F-mediated membrane fusion and have important implications for the identification of therapeutic strategies and vaccines targeting RSV F protein conformational changes.

3-O-3′(or 2′)-Methylsuccinyl-betulinic acid (MSB) derivatives were separated by using recycle HPLC. The structures of four isomers were assigned by NMR and asymmetric synthesis. 3-O-3′S-Methylsuccinyl-betulinic acid (3′S-MSB, 4) exhibited potent anti-HIV activity with an EC50 value of 0.0087 μM and a TI value of 6.3 × 103, which is comparable to the data for bevirimat (DSB, PA-457), a current clinical trials drug that was also derived from betulinic acid. The anti-HIV potency of 4 was slightly better than that of AZT.

3′,4′-Di-O-(−)-camphanoyl-(+)-cis-khellactone (DCK, 1) demonstrated extremely potent inhibitory activity against HIV-1 replication in H9 lymphocytic cells with an EC50 value of 2.56 × 10−4 μM and a therapeutic index (TI) of 1.37 × 105 in our prior research.1 In subsequent structural modification studies, numerous DCK derivatives were synthesized and at least 20 DCK analogs have shown promising inhibitory activity against HIV-1 replication in H9 lymphocytes.2 Among them, 3-methyl, 4-methyl, and 5-methyl substituted DCKs were much more potent than DCK and AZT in the same assay with EC50 and TI values ranging from 5.25 × 10−5 to 2.39 × 10−7 μM and 2.15 × 106 to 3.97 × 108, respectively.3 In addition, a preliminary mechanistic study showed that 3-hydroxymethyl-4-methyl DCK inhibits HIV reverse transcriptase (RT) via a different mechanism of action from those of current clinical anti-HIV/AIDS drugs.4 It was also found that DCK analogs are strongly synergistic with approved drugs such as AZT and act at a point in the virus life cycle immediately following the target for AZT and nevirapine.4 In our recent research on structural modification of 4-methyl DCK (2), the ring oxygen atom in the A or C ring of DCK was replaced by a sulfur atom, and these sulfur-containing analogs also exhibited potent inhibitory effects on HIV-1 replication in H9 lymphocytes.5,6 Moreover, gem-dimethyl substitution at the 8-position was found to be preferable to larger alkyl substituents or hydrogen atoms.7 In a continuing effort to identify the pharmacophores in this class of potent anti-HIV agents, we designed a new series of DCK analogs, namely 7-carbon-DCK derivatives (3a–c). In these compounds, a methylene group replaces the oxygen in the C ring of DCK. Thus, these analogs are bioisosteres of DCK, and the effect of the 7-oxygen atom on the anti-HIV activity of DCK-type compounds can be further explored. In addition, to help determine the possible impact of the 8,8-dimethyl groups, both unsubstituted (3a–b) and dimethylated (3c) analogs were prepared. Herein, we report the synthesis of compounds 3a–c and their preliminary anti-HIV bioassay results (Fig. 1). Figure 1 Structures of DCK (1), 4-methyl DCK (2) and 7-carbon-DCK analogs (3a–c). The synthesis of 3a–b was accomplished by a 7-step sequence, as illustrated in Scheme 1. The key intermediates 7,8-dihydro-benzo[h]chromen-2-one (10a) and its 4-methyl analog (10b) were prepared according to the procedure reported in our prior work.8 Sharpless asymmetric dihydroxylation (AD) of 10a and 10b afforded dihydroxy derivatives 11a and 11b in moderate yield (45–49%).9 Finally, 7-carbon-DCK analogs 3a and 3b were obtained in 62% and 82% yields, respectively, by acylation of 11a and 11b with (S)-(−)-camphanic chloride in CH2Cl2 at room temperature with pyridine as acid scavenger. Scheme 1 Synthesis of 3c: (i) t-BuOK/THF, reflux, 5 h, CH3I, 0.5 h; (ii) H2, Pd-C, CH3SO3H, HOAc, CH3COOC2H5, C2H5OH, rt, 36 h; (iii) BBr3/CH2Cl2, −78°C; (iv) CH3COCH2COOC2H5, POCl3, Benzene, reflux, 24 h, 66.0%; (v) CrO3, HOAc, rt, 30 h, (yield: ... As shown in Scheme 2, the preparation of 3c followed a slightly different synthetic route with 5-methoxy-1-tetralone (12) as starting material. Dimethylation of 12 with CH3I in the presence of t-BuOK afforded 2,2-dimethyl-5-methoxy-1-tetralone (13) in 91% yield.10 Reduction of dimethylated tetralone 13 with H2 catalyzed with 10% Pd-C gave 1,2,3,4-tetrahydro-5-methoxy-2,2-dimethylnaphthalene (14) quantitatively.11 Demethylation of 14 with BBr3 resulted in the formation of phenol derivative 15 in 98% yield.11 The remaining synthetic steps followed those detailed above for 3a–b from phenol 5. The target compound 3c was thus obtained in an overall yield of 5% via a six-step reaction sequence starting from 15. Scheme 2 Synthesis of 3a–b: (i) Raney Ni-Al alloy, 1% aq. KOH/water, 90°C, 2 h; (ii) L-Malic acid, H2SO4, HOAc, 140°C, 6 h (R=H); CH3COCH2COOC2H5, POCl3, 100°C, 18 h (R=CH3); (iii) CrO3, HOAc, rt, 3 days; (iv) NaBH4, CH3OH, 1 h; ... The anti-HIV activities of compounds 3a–c were evaluated in H9 lymphocytes, with AZT as the reference compound. The bioassay data are shown in Table 1 and indicated that all three compounds inhibited HIV replication and had reasonable therapeutic index (TI) values. Compounds 3a and 3b had significant EC50 values of 0.068 and 0.083 μM, respectively. Thus, the presence of the C-4 methyl in these 7-carbon DCK analogs did not lead to increased potency, in contrast to results with DCK and 4-methyl DCK. Although an absence of gem-dimethylation was detrimental in the 7-oxy DCK series,7 it was hard to make a definitive conclusion in the 7-carbon DCK series (comparison of 3c with 3b) due to solubility problems with 3c. However, the 7-carbon analog 3b was more potent and had a higher TI than the corresponding demethylated 7-oxy DCK derivative, 2′,2′-dihydro-4-methyl DCK (EC50 = 6.9 μM, TI > 6).7 Compound 3b was also more potent against HIV replication but was more cytotoxic than the analogous 7-thio analog (EC50 = 0.141 μM, TI = 1,110).6 Table 1 Anti-HIV data of compounds 3a–c in acutely infected H9 lymphocytes Further structural modification and biological screening are in progress as these promising bioassay results demonstrate that 7-carbon-DCK analogs merit attention as potential HIV-1 inhibitors.

3-O-(3′,3′-dimethylsuccinyl) betulinic acid, also termed PA-457 or DSB, is a novel HIV-1 inhibitor that blocks virus maturation by disrupting cleavage of the capsid precursor, CA-SP1. To better define the molecular target for PA-457, we prepared a panel of mutant viruses with point deletions spanning the CA-SP1 cleavage domain and characterized each of these viruses for PA-457 sensitivity. Our results indicate that amino acid residues in the N-terminal half of SP1 serve as determinants of PA-457 activity, while residues in the C-terminal half of SP1 were not involved in compound activity. These findings support and extend previous observations that PA-457 is a specific inhibitor of CA-SP1 cleavage and identify the CA-SP1 domain as the primary viral determinant for this novel inhibitor of HIV-1 replication.

Bioactivity-directed fractionation of an ethanolic extract of the fruits of Schisandra rubriflora led to the isolation and identification of dibenzocyclooctadiene lignans including the new lignans rubrisandrins A (1a + 1b) and B (2) and the known lignans gomisin J (3), (+/-)-gomisin M1 (4), (+)-gomisin M2 (5), schisanhenol (6), deoxyschisandrin, schisantherin B, schisandrin, tigloylgomisin P, gomisin O, angeloylgomisin P, and epigomisin O. Their structure and stereochemistry were determined by spectroscopic methods, including 2D-NMR techniques. Compounds 1 and 3-6 were active as anti-HIV agents. (+/-)-Gomisin M1 (4) exhibited the most potent anti-HIV activity, with EC50 and therapeutic index (TI) values of0.65 microM and68, respectively.

New HIV therapies are urgently needed to address the growing problem of drug resistance. In this article, we characterize the anti-HIV drug candidate 3- O -(3′,3′-dimethylsuccinyl) betulinic acid (PA-457). We show that PA-457 potently inhibits replication of both WT and drug-resistant HIV-1 isolates and demonstrate that the compound acts by disrupting a late step in Gag processing involving conversion of the capsid precursor (p25) to mature capsid protein (p24). We find that virions from PA-457-treated cultures are noninfectious and exhibit an aberrant particle morphology characterized by a spherical, acentric core and a crescent-shaped, electron-dense shell lying just inside the viral membrane. To identify the determinants of compound activity we selected for PA-457-resistant virus in vitro . Consistent with the effect on Gag processing, we found that mutations conferring resistance to PA-457 map to the p25 to p24 cleavage site. PA-457 represents a unique class of anti-HIV compounds termed maturation inhibitors that exploit a previously unidentified viral target, providing additional opportunities for HIV drug discovery.

Twenty-six semisynthetic ent-kaurane derivatives of linearol (1) have been investigated for their anti-HIV effects. Five compounds (4, 7, 11, 25, and 26) showed significant activity against HIV replication in H9 lymphocyte cells with EC(50) values in the range

A new triterpenoid named melliferone (1), three known triterpenoids, moronic acid (2), anwuweizonic acid (3), and betulonic acid (4), and four known aromatic compounds (5-8) were isolated from Brazilian propolis and tested for anti-HIV activity in H9 lymphocytes. Moronic acid (2) showed significant anti-HIV activity (EC(50) 186) and was modified to develop more potent anti-AIDS agents.

Ru(II)/Ru(III) polypyridyl complexes containing 2,6-(2'-benzimidazolyl)-pyridine or chalcone as co-ligands were synthesized and characterized previously (Mishra, L.; Sinha, R. Indian J. Chem., Sec. A 2001, in press. Mishra, L.; Sinha, R. Indian J. Chem., Sec. A, 39A, 2000, 1131). Their interaction with aqueous buffered calf thymus DNA was measured. (Novakova, O.; Kasparkova, J.; Vrana, O.; van Vliet, P. M., Reedijk, J.; Brabec, V., Biochem. 34, 1995, 12369 and these results prompted additional screening for anti-HIV (human immunodeficiency virus) activity against DNA replication in H9 lymphocytes and cytotoxic activity against eight tumor cell lines. The most active compounds were 17 in the former assay (EC(50)0.1 microg/mL and TI23.1) and 3, 8, 10, and 14 in the latter assay, especially selectively against the 1A9 ovarian cancer cell line (IC(50) = 4.1, 3.8, 3.6, and 2.5 microg/mL, respectively).

Six 3-substituted 3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone derivatives (3–8) were synthesized from 3-methyl DCK (2). 3-Hydroxymethyl DCK (6) exhibited potent anti-HIV activity in H9 lymphocytes with EC50 and TI values of 1.87×10−4 μM and 1.89×105, respectively. These values are similar to those of DCK and better than those of AZT in the same assay.

3-O-Acyl-betulin and -dihydrobetulin derivatives were prepared and evaluated for anti-HIV activity. 3-O-Glutaryl-dihydrobetulin (17) demonstrated extremely potent anti-HIV activity with an EC(50) value of 2 x 10(-5) microM and a TI value of 1.12 x 10(6). 3-O-(3',3'-Dimethylsuccinyl)- and 3-O-(3',3'-dimethylglutaryl)-dihydrobetulins (15, 16) were also potent anti-HIV compounds with EC(50) values of 0.0017 and 0.0013 microM, respectively, and TI values of 16,160 and 19,530, respectively.

Two thia-DCK analogs (3a,b) were synthesized and evaluated for inhibition of HIV-1 replication in H9 lymphocytes. Compound 3a showed potent anti-HIV activity with an EC50 value of 0.14 microM and a therapeutic index of 1110. However, the corresponding 6-tert-butyl-substituted compound (3b) showed no suppression. The bioassay results indicated that thia-DCK analogs merit attention as potential HIV-1 inhibitors.

Two 1-thia-DCK analogues ( 9a and 9b ) were synthesized and evaluated for inhibition of HIV-1 replication in H9 lymphocytes. Compound 9a showed excellent anti-HIV activity with an EC 50 value of 0.00012 μM and therapeutic index of 1,408,000. Compound 9b was less active with EC 50 and TI values of 3.11 μM and 62.3, respectively. The bioassay results indicated that thia-DCK analogues merit attention as potential HIV-1 inhibitors.

While it has been established that peptides modeling the C-helical region of human immunodeficiency virus type 1 gp41 are potent in vivo inhibitors of virus replication, their mechanism of action has yet to be determined. It has been proposed, but never directly demonstrated, that these peptides block virus entry by interacting with gp41 to disrupt the formation or function of a six-helix bundle structure. Using a six-helix bundle-specific monoclonal antibody with isolate-restricted Env reactivity, we provide the first direct evidence that, in receptor-activated viral Env, C-peptide entry inhibitors bind to the gp41 N-helical coiled-coil to form a peptide/protein hybrid structure and, in doing so, disrupt native six-helix bundle formation.

Six 3-substituted 3',4'-di-O-(S)-camphanoyl-(+)-cis-khellactone derivatives (3-8) were synthesized from 3-methyl DCK (2). 3-Hydroxymethyl DCK (6) exhibited potent anti-HIV activity in H9 lymphocytes with EC(50) and TI values of 1.87 x 10(-4) microM and 1.89 x 10(5), respectively. These values are similar to those of DCK and better than those of AZT in the same assay.