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Virology 417 (2011) 253–258

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Virology

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Rapid Communication Synergistic activity profile of griffithsin in combination with tenofovir, maraviroc and enfuvirtide against HIV-1 clade C

Geoffrey Férir a, Kenneth E. Palmer b, Dominique Schols a,⁎

a Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium b Department of Pharmacology and Toxicology and Owensboro Cancer Research Program, James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, USA

article info abstract

Article history: Griffithsin (GRFT) is possibly the most potent anti-HIV peptide found in natural sources. Due to its potent and Received 10 May 2011 broad-spectrum antiviral activity and unique safety profile it has great potential as topical microbicide Returned to author for revision 6 June 2011 component. Here, we evaluated various combinations of GRFT against HIV-1 clade B and clade C isolates in Accepted 7 July 2011 primary peripheral blood mononuclear cells (PBMCs) and in CD4+ MT-4 cells. In all combinations tested, Available online 28 July 2011 GRFT showed synergistic activity profile with tenofovir, maraviroc and enfuvirtide based on the median effect principle with combination indices (CI) varying between 0.34 and 0.79 at the calculated EC level. Keywords: 95 Griffithsin Furthermore, the different glycosylation patterns on the viral envelope of clade B and clade C gp120 had no Tenofovir observable effect on the synergistic interactions. Maraviroc Overall, we can conclude that the evaluated two-drug combination increases their antiviral potency and Enfuvirtide supports further clinical investigations in pre-exposure prophylaxis for GRFT combinations in the context of Synergy HIV-1 clade C infection. Combination index © 2011 Elsevier Inc. All rights reserved. Microbicide HIV-1 clade C Glycosylation PBMCs

Introduction 1% tenofovir gel product showed partial efficacy in prevention of HIV- 1 transmission (Abdool Karim et al., 2010). As for systemic HIV The broad genetic diversity of human immunodeficiency viruses treatment, the most effective HIV prophylaxis strategy will involve makes it very difficult to generate an effective vaccine (McElrath and combinations of various antiretroviral drugs. Viral entry is considered Haynes, 2010). While HIV-1 clade C dominates the HIV-1 epidemic an important target in combating HIV/AIDS infections and thus entry worldwide with N50% of the total viral infections, predominately in inhibitors are proposed as candidates for microbicidal applications. Southern Africa, India and other parts of Asia, HIV-1 subtype B is The gp41 fusion peptide inhibitor enfuvirtide (Fuzeon) was the first mainly found in (Western-) Europe and in the Americas, accounting clinically approved entry inhibitor and has clearly proven its around 10% of the total infections (Buonaguro et al., 2007). In areas effectiveness (Kilby et al., 1998; Kilby et al., 2002). The only HIV co- where they have been recently introduced, like Brazil, Clade C viruses receptor inhibitor, the CCR5 antagonist maraviroc (Selzentry), was become rapidly established, and have started to replace the previously approved for the treatment of HIV infections, in combination with dominant Clade B epidemic (de Oliveira et al., 2010). The standard other antiretroviral drugs (MacArthur and Novak, 2008). combination antiretroviral therapy does not cure HIV, making At present, griffithsin (GRFT) is possibly the most potent and prevention of novel infections of extreme importance. A high number broad-spectrum HIV entry inhibitor as it inhibits viral replication at of the new HIV infections occur through heterosexual intercourse. pM levels by specifically targeting multiple mannose-rich residues Therefore, self-administered topical microbicides (e.g. vaginal gel or present on the viral envelope protein gp120 (Emau et al., 2007; Mori intravaginal ring devices) could be very helpful tools to reduce the et al., 2005). GRFT has a molecular weight of 12.7 kDa and was infection rates. In the last decade, many microbicidal clinical trials originally isolated from the red alga Griffithsia sp. (Mori et al., 2005). It failed due to safety problems or lack of efficacy (Lacey et al., 2010; contains 121 amino acids and has a unique dimeric structure McCormack et al., 2010; Van Damme et al., 2008). However, recently a (Ziółkowska et al., 2006). Production of recombinant forms of GRFT in E. coli and in the Nicotiana benthamiana plant did not alter its potent

anti-HIV activity (IC50: 0.043–0.63 nM) nor its safety profile com- ⁎ Corresponding author. Fax: +32 16 337340. pared with native isolated GRFT (CC : N1000 nM) (Mori et al., 2005; E-mail addresses: [email protected] (G. Férir), 50 [email protected] (K.E. Palmer), [email protected] O'Keefe et al., 2009). Besides inhibiting HIV-1 subtype B strains, GRFT (D. Schols). was also able to potently inhibit HIV-1 clade A and clade C viruses

0042-6822/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2011.07.004 254 G. Férir et al. / Virology 417 (2011) 253–258

Table 1

EC50 values of GRFT (nM), tenofovir (μM), maraviroc (nM) and enfuvirtide (nM) alone and in combination at equipotent (1:1) ratio against R5 HIV-1 in PBMCs.

a b EC50 individual drug EC50 combination GRFT Inhibitor GRFT Inhibitor ⁎ Clade B/BaL Tenofovir 0.16±0.02 1.3±0.9 0.05±0.02 0.64±0.19 ⁎ Maraviroc 0.17±0.02 6.6±1.9 0.05±0.01 2.3±0.6 ⁎ ⁎ Enfuvirtide 0.21±0.01 41.0±7.3 0.06±0.01 5.6±1.6 ⁎ Clade C/DJ259 Tenofovir 0.11±0.01 0.95±0.19 0.07±0.01 0.84±0.03 Maraviroc 0.34±0.30 1.6±0.9 0.03±0.01 0.48±0.14 ⁎ Clade C/ZAM18 Tenofovir 0.61±0.01 0.53±0.37 0.12±0.08 0.27±0.11 Maraviroc 1.8±0.6 4.4±1.7 0.57±0.30 3.4±1.4 ⁎ Clade C/ETH2220 Tenofovir 0.59±0.06 0.72±0.11 0.05±0.01 0.59±0.03 ⁎ Maraviroc 0.69±0.19 3.1±1.5 0.13±0.02 2.1±0.4

a 50% effective concentration for HIV-1 infection in PBMCs after single drug treatment. Mean EC50s±SEM of 2–4 donors are shown. b 50% effective concentration after two-drug combination at equipotent ratio (1:1). ⁎ pb0.05 (unpaired T-test), compared to single drug treatment. isolated from blood and cervico-vaginal samples (Alexandre et al., 2010; O'Keefe et al., 2009). Glycosylation patterns between different HIV-1 clades vary (Zhang et al., 2004) and thus play an important role in the antiviral activity of carbohydrate-binding agents (CBAs). A Molecular studies with GRFT have shown that introduction of the glycosylation sites N234 and N295 in HIV-1 clade C virus increased its potency (Alexandre et al., 2010). Here, we describe that GRFT acts synergistically with tenofovir, maraviroc and enfuvirtide in PBMCs against R5 HIV-1 clade B BaL. We observed synergistic effects with the combination of GRFT/tenofovir and GRFT/maraviroc on 3 different R5 clade C HIV-1 clinical isolates (DJ259, ZAM18 and ETH2220) with different glycosylation patterns. Additionally, synergistic interactions were also observed in MT-4 cells against X4 HIV-1 NL4.3 with GRFT/tenofovir, GRFT/AMD3100 and GRFT/enfuvirtide. These data show that combination of GRFT with tenofovir, maraviroc or enfuvirtide increases antiviral potency and support further clinical research on these combinations as potential microbicide also in the context of HIV-1 clade C infections. B Results

GRFT is synergistic with tenofovir, maraviroc and enfuvirtide against clade B HIV-1 R5 strain BaL in PBMCs

First, we determined the 50% effective concentrations (EC50s) of GRFT, tenofovir, maraviroc and enfuvirtide alone, followed by the two-drug combinations GRFT/tenofovir, GRFT/maraviroc and GRFT/ enfuvirtide against HIV-1 R5 clade B BaL replication. The EC50s of each inhibitor alone and in combination are shown in Table 1. Overall, GRFT inhibits HIV-1 BaL with an average EC50 of 0.18 nM and significant reductions (pb0.05)inGRFTconcentrationswerenotedafter combination with tenofovir (p=0.048), maraviroc (p=0.0094) and C enfuvirtide (pb0.0001). A statistical significant difference was also calculated for enfuvirtide in combination with GRFT (p=0.0032) using the unpaired T-test when compared to single drug treatment (Table 1). As shown in Fig. 1, combination of GRFT with each inhibitor tested, resulted in a more efficient inhibitory profile against HIV-1 replication as measured by p24 HIV-1 core Ag ELISA. Combination indices (CI) were determined to evaluate the type of drug–drug interactions, where CIb0.9 are synergistic, 0.9bCIb1.1 are additive effects and CIN1.1 are antagonistic. The obtained CI values varied between 0.44 and 0.53, resulting thus in synergy at the calculated EC95 level for each combination (Table 2).

Synergistic anti-HIV-1 activity of GRFT with tenofovir and maraviroc against various clade C clinical isolates in PBMCs Fig. 1. The activity profile against R5 HIV-1 BaL replication (% virus production measured by p24 HIV-1 core Ag ELISA 10 days post-infection) in PBMCs of GRFT alone The number of clade C HIV-1 infections dominates the number of or combined with tenofovir (A), or with maraviroc (B) or with enfuvirtide (C). A clade B infections in Africa. Consequently, an effective microbicide must representative donor is shown from 4 different PBMCs donor experiments. G. Férir et al. / Virology 417 (2011) 253–258 255

Table 2 Table 4

Combination index determination for various GRFT combinations against clade B BaL in EC50 values of GRFT (nM), tenofovir (μM), enfuvirtide (nM) and AMD3100 (nM) alone PBMCs. and in combination at equipotent (1:1) ratio against HIV-1 NL4.3 in MT-4 cells.

a b HIV-1 strain BaL (clade B) EC50 individual drug EC50 combination Combination CIa Synergyb GRFT Inhibitor GRFT Inhibitor ⁎ ⁎ GRFT/tenofovir 0.47±0.05 +++ Tenofovir 0.10±0.02 6.3±0.6 0.029±0.006 1.9±0.5 ⁎ ⁎ GRFT/maraviroc 0.44±0.05 +++ Enfuvirtide 0.12±0.02 23.7±5.5 0.015±0.003 4.9±0.7 ⁎ ⁎ GRFT/enfuvirtide 0.53±0.05 +++ AMD3100 0.13±0.02 16.0±1.7 0.044±0.004 5.8±1.3

a a Combination index (CI) calculated at the EC95-level. CIb0.9 indicates synergism; EC50 concentrations for individual drug treatment against NL4.3 infection in MT-4 0.9bCIb1.1 indicates additive effects and CIN1.1 indicates antagonism. Mean CI±SEM cells. Mean values±SEM from 4 independent experiments are shown. b are shown. EC50 concentrations (mean±SEM) after two-drug combinations at equipotent b Synergy level: 0.85bCIb0.9: + (slight synergism); 0.7bCIb0.85: ++ (moderate ratio (1:1) from 4 individual experiments are shown. synergism); 0.3bCIb0.7: +++ (synergism); 0.1bCIb0.3: ++++ (potent ⁎ pb0.05 (according to unpaired T-Test). synergism).

Table 3 Stronger synergy was observed against ZAM18 and ETH2220 with CI Synergy calculations against various clade C HIV-1 clinical isolates after GRFT/tenofovir values of 0.51±0.14 and 0.59±0.24, respectively (Table 3 and Fig. 2). and GRFT/maraviroc combination treatment.

HIV strains Synergya GRFT is synergistic with tenofovir, enfuvirtide and AMD3100 against X4 NL4.3 in MT-4 cells Combi DJ259 ZAM18 ETH2220

GRFT/tenofovir ++(+) ++ ++ We further evaluated if synergistic interactions occur when GRFT GRFT/maraviroc +++ +++ +++ was combined with tenofovir, enfuvirtide and the specific CXCR4 a Synergy level: 0.85bCIb0.9: + (slight synergism); 0.7bCIb0.85: ++ (moderate antagonist AMD3100 on MT-4 cells using the laboratory-adapted HIV-1 b b b b synergism); 0.3 CI 0.7: +++ (synergism); 0.1 CI 0.3: ++++ (potent X4 strain NL4.3. The EC values for each drug alone and in synergism). 50 combination are shown in Table 4. After combination, GRFT effective concentrations decreased significantly in each combination tested (pb0.05) and a similar observation was noted for each combined inhibit many subtypes of HIV-1. Therefore, we investigated combina- inhibitor (pb0.05; Table 4). As seen with the R5 HIV-1 strains, all tions of GRFT with tenofovir and maraviroc against three clinical HIV-1 combinations increase the antiviral potency of GRFT against the X4 isolates with different glycosylation patterns. The susceptibility of GRFT, virus. The strongest synergistic interaction was observed between tenofovir and maraviroc, alone and their combinations against these GRFT and enfuvirtide (CI, 0.34±0.10; Table 5). A comparable clade C viruses is shown in Table 1.Significant reductions were observed synergistic profile was observed with the combinations GRFT/ for the GRFT 50% inhibitory dose in each combination with tenofovir tenofovir and GRFT/AMD3100 with a CI of 0.60 ±0.03 and 0.64 ±

(pb0.05) (Table 1). With maraviroc, the EC50 for GRFT decreased 0.06, respectively (Table 5). significantly against ETH2220 (p=0.0405). It is important to note that no large differences were observed in anti-HIV activity in PBMCs against Discussion clade C viruses compared with clade B (Table 1). Overall, we can conclude that combination of GRFT with tenofovir or maraviroc The proof-of-concept trial of a 1% tenofovir vaginal gel product increases its antiviral potency (Table 1). CI calculations showed synergy (CAPRISA 004) for the prevention of HIV infection in women opened between GRFT and tenofovir against the 3 isolates tested: DJ259 (CI, novel perspectives in the field of microbicidal research (Abdool Karim 0.70±0.10), ZAM18 (CI, 0.76±0.10) and ETH2220 (CI, 0.79±0.02) et al., 2010). However, like systemic treatment of HIV/AIDS infections, (Table 3 and Fig. 2). A comparable synergistic interaction was also found single drug microbicides are perhaps not potent enough and could after combination with maraviroc against DJ259 (CI, 0.67±0.08). also increase the risk of transmission of resistant viruses, making combinations of at least 2 different antiretroviral drugs with broad- spectrum anti-HIV activity the best prevention strategy against viral transmission Klasse et al. (2008). An effective microbicide must also have a good cervicovaginal safety and pharmacokinetic/dynamic profile (Doncel and Clark, 2010). Griffithsin is not mitogenic and showed no toxicity in rabbit vaginal irritation tests as well as no induction of pro-inflammatory cytokines in human cervical explants

Table 5

Combination indices at the EC95 level of GRFT combinations in two-drug assays against X4 HIV-1 NL4.3.

GRFT in combination with

Tenofovir AMD3100 Enfuvirtide

Combination indexa 0.60±0.03 0.64±0.06 0.34±0.10 Synergyb +++ +++ +++

a Combination index (CI) calculated at the EC95 level represented by the mean value± SEM from 4 independent experiments in MT-4 cells infected with HIV-1 NL4.3. CIb0.9 indicates synergism; 0.9 bCIb1.1 indicates additive effects and CI N1.1 indicates antagonism. Fig. 2. Mean combination indices (±SEM of 3–4 different PBMC donors) of GRFT/ b Synergy level: 0.85bCIb0.9: + (slight synergism); 0.7bCIb0.85: ++ (moderate tenofovir and GRFT/maraviroc determined against clade C R5 HIV-1 isolates DJ259, synergism); 0.3bCIb0.7: +++ (synergism); 0.1bCIb0.3: ++++ (potent ZAM18 and ETH2220. synergism). 256 G. Férir et al. / Virology 417 (2011) 253–258 at 2 μM(O'Keefe et al., 2009). We also found no induction of any making a GRFT/maraviroc containing gel or controlled-release cytokines nor cellular activation markers in human PBMCs at 2 μM intravaginal ring device good candidates for HIV prevention studies. using the Bio-plex Human Cytokine 27-plex assay (unpublished data). Although sexual transmission of HIV mainly occurs by R5 viruses, GRFT belongs to the class of carbohydrate-binding agents (CBAs) presumably because of lower replication “fitness” of X4 viruses, we and is, to our knowledge, the most potent CBA described for investigated potential synergistic interactions against X4 viruses. A inhibiting HIV infection and replication in the pM range (Alexandre study with macaques showed that X4 viruses can also be transmitted et al., 2010; Mori et al., 2005; O'Keefe et al., 2009). CBAs are vaginally (Miller and Hu, 1999). We used CD4+ MT-4 cells, which are attractive microbicide candidatesastheytargetHIVat4different CCR5− and CXCR4+, therefore in these experiments maraviroc was intervention steps, namely (i) inhibition of cell-free virus replication replaced by the specific CXCR4 antagonist AMD3100 (Schols et al., and (ii) inhibition of syncytium formation between uninfected 1997). GRFT showed synergistic interactions in all combinations CD4+ T cells and HIV-infected T cells, (iii) inhibition of capture by tested against X4 HIV-1 NL4.3 (Table 5). Potent synergistic in- DC-SIGN and (iv) transmission of captured virus to uninfected CD4+ T teractions between enfuvirtide/AMD3100 in PBMCs were also cells (Balzarini, 2007). observed by other research groups (Tremblay et al., 2000). Certain sexually transmitted diseases (such as Chlamydia or Herpes Enfuvirtide showed potent anti-HIV activity, a safe pharmacoki- Simplex Virus) can increase the risk on HIV transmission (Abu-Raddad netic profile and became the first approved peptidic entry inhibitor for et al., 2008; Baeten et al., 2007). The results of the CAPRISA 004 trial the treatment of HIV/AIDS infections (Kilby et al., 1998, 2002). showed that tenofovir also reduced the genital herpes infections Enfuvirtide is mainly used in non-African countries, so we only (Abdool Karim et al., 2010). In addition to its activity against HIV-1, evaluated the combination with GRFT against HIV-1 BaL. Here we also GRFT is also active against HSV-2 (Palmer et al., manuscript in demonstrated that GRFT has synergistic interactions with enfuvirtide preparation). Given the established efficacy of tenofovir vaginal gel against HIV-1 clade B. Since its discovery, novel derivatives of against HSV-2 acquisition, a GRFT/tenofovir combination microbicide enfuvirtide have been generated (such as T-1249 and T-2635). An becomes an appealing product to combat HIV transmission. advantage of these fusion inhibitors compared with co-receptor Here, we showed that the potent anti-HIV CBA GRFT can be antagonists is their tropism independent antiviral activity. Macaques combined with the nucleotide reverse transcriptase inhibitor (RTI), that received a T-1249-containing vaginal gel showed inhibition of tenofovir, the CCR5 HIV co-receptor antagonist, maraviroc and the SHIV replication (Veazey et al., 2008) and this peptide showed a gp41 fusion inhibitor enfuvirtide and that all combinations are superior antiviral profile compared with enfuvirtide. Resistance synergistic against clade B (mainly present in the Americas, analysis of this second generation peptide revealed that mutations Australasia and Europe) and clade C, which accounts for approxi- occur at position 38 (V38) in gp41 and resulted in enfuvirtide cross- mately 60% of the HIV-1 infections, worldwide (mainly in Africa and resistance. However, a third generation peptide, T-2635, retained its the Indian subcontinent, with expanding epidemics elsewhere). By antiviral activity (Eggink et al., 2008). These data and our observations combining GRFT with an entry inhibitor targeting ensuing steps in the show that enfuvirtide (and its later generation peptides) could also be entry process or with a RTI, a broader range of (multi-drug resistant) useful in the prevention of HIV transmission. HIV subtypes can be blocked. Overall, due to its potent and broad-spectrum anti-HIV activity as GRFT acts as a virucide by targeting multiple glycans present on well as its synergistic interactions with tenofovir, maraviroc and gp120 (Mori et al., 2005). Alexandre et al. (2010) showed that the N- enfuvirtide, we believe that GRFT has a great potential as microbicide glycosylations on position 295 (N295) and 234 (N234) are crucial in the prevention of sexual transmission of HIV when co-formulated binding sites for GRFT and their absence decreased its antiviral in appropriate gel or solid dosage form, or in a sustained-release potency. The HIV-1 strains NL4.3 (clade B) and DJ259 (clade C) both intravaginal ring device. All GRFT-antiretroviral combinations tested contain these N-linked glycosylations. BaL (clade B) lacks N234, showed a synergistic profile, with improved antiviral potency. The ZAM18 (clade C) lacks N295 and ETH2220 (clade C) lacks both N- observed synergy for the GRFT/enfuvirtide and the GRFT/maraviroc glycosylations. The complete amino acid sequences are available in combinations may be a reflection of their common target, viral entry, GenBank with GenBank ID: AAB60578, BAH97458, ABC55874, but the agents are targeting subsequent steps during the entry BAH97494 and Q75008 resp. (www.ncbi.nlm.nih.gov/protein). In process. As we showed previously with several other members of the our experiments performed in PBMCs, GRFT lost almost no activity CBA family (e.g. microvirin and Hippeastrum Hybrid agglutinin), also against the three different clade C isolates compared to the well- GRFT has no negative effects on the cellular uptake and processing of described and widely used laboratory strain clade B BaL (Table 1). the RTI tenofovir. Synergy may enable reductions in effective dose of A strong hallmark of the class of CBAs is their high genetic barrier each component, with corresponding decreases in side-effect profiles, to resistance and excellent physicochemical properties (e.g. resistant and even reduction in the product costs. Our data strongly support to high temperature and low pH) (Balzarini et al., 2004; Huskens et al., further translation research on these GRFT-antiretroviral combina- 2010). Griffithsin maintained its potent antiviral activity in both acidic tions as potential microbicides. and alkaline pH (Emau et al., 2007) and we recently showed that tenofovir can be combined with various other CBAs in vitro against R5 Materials and methods and X4 HIV-1 (Férir et al., 2011). In vivo studies using a tenofovir/GRFT gel combination are planned to evaluate various antiviral efficacy Cells and cell culture parameters in preparation for human clinical studies. Maraviroc is a specific CCR5 interacting small chemical molecule MT-4 cells were a gift from Dr. L. Montagnier (at that time at the with potent activity against R5 viruses (Dorr et al., 2005). For systemic pasteur institute, Paris, France) and cultured in RPMI-1640 medium HIV treatment, maraviroc is used in combination with other containing 10% FCS (Hyclone, Perbio Science, Aalst, Belgium) and antiretroviral drugs and various studies have shown that it also has 2mML-glutamine (Invitrogen, Merelbeke, Belgium) at 37 °C and 5% potential for microbicidal applications. An open-labeled pharmacoki- CO2. Peripheral blood mononuclear cells (PBMCs) from healthy netical study in healthy women showed that orally administered donors were isolated out of a buffy coat obtained from the Blood maraviroc concentrates in the female genital tract (Dumond et al., Transfusion Center (U.Z. Leuven, Belgium). PBMCs were also cultured 2009). A recent study using maraviroc in a gel-based formulation RPMI-1640 supplemented with 10% FCS, 2 mM L-glutamine and protects rhesus macaques from infection with CCR5-tropic SHIV 2 ng/ml IL-2 (Roche Molecular Biochemicals, Indianapolis, USA) and (Veazey et al., 2010). GRFT shows synergistic interactions with activated with 2 μg/ml PHA (Sigma-Aldrich, Bornem, Belgium) for maraviroc against clade B and clade C HIV-1 strains (Tables 2 and 3), 3 days before antiviral testing. G. Férir et al. / Virology 417 (2011) 253–258 257

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