PMID: 

J Control Release. 2020 May 10 ;321:84-99. Epub 2020 Feb 5. PMID: 32035194

Abstract Title: 

Sustained-release Griffithsin nanoparticle-fiber composites against HIV-1 and HSV-2 infections.

Abstract: 

Human immunodeficiency virus (HIV-1) and herpes simplex virus 2 (HSV-2) affect hundreds of millions of people worldwide. The antiviral lectin, Griffithsin (GRFT), has been shown to be both safe and efficacious against HSV-2 and HIV-1 infections in vivo. The goal of this work was to develop a multilayered nanoparticle (NP)-electrospun fiber (EF) composite to provide sustained-release of GRFT, and to examine its safety and efficacy in a murine model of lethal HSV-2 infection. Composites were fabricated from polycaprolactone (PCL) fibers surrounding polyethylene oxide (PEO) fibers that incorporated methoxy poly(ethylene glycol)-b-poly(lactide-co-glycolide) (mPEG-PLGA) GRFT NPs. GRFT loading and release were determined via ELISA, showing that NP-EF composites achieved high GRFT loading, and provided sustained-release of GRFT for up to 90 d. The in vitro efficacy of GRFT NP-EFs was assessed using HIV-1 pseudovirus assays, demonstrating complete in vitro protection against HIV-1 infection. Additionally, sustained-release NP-EFs, administered 24 h prior to infection, prevented against a lethal dose of HSV-2 infection in a murine model. In parallel, histology and cytokine expression from murine reproductive tracts and vaginal lavages collected 24 and 72 h post-administration were similar to untreated mice, suggesting that NP-EF compositesmay be a promising and safe sustained-delivery platform to prevent HSV-2 infection. Future work will evaluate the ability to provide prolonged protection against multiple virus challenges, and different administration times with respect to infection.

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PMID: 

Viruses. 2016 11 17 ;8(11). Epub 2016 Nov 17. PMID: 27869695

Abstract Title: 

Studies in a Murine Model Confirm the Safety of Griffithsin and Advocate Its Further Development as a Microbicide Targeting HIV-1 and Other Enveloped Viruses.

Abstract: 

Griffithsin (GRFT), a lectin fromspecies, inhibits human immunodeficiency virus-1 (HIV-1) replication at sub-nanomolar concentrations, with limited cellular toxicity. However, in vivo safety of GRFT is not fully understood, especially following parenteral administration. We first assessed GRFT's effects in vitro, on mouse peripheral blood mononuclear cell (mPBMC) viability, mitogenicity, and activation using flow-cytometry, as well as cytokine secretion through enzyme-linked immunosorbent assay (ELISA). Toxicological properties of GRFT were determined after a single subcutaneous administration of 50 mg/kg or 14 daily doses of 10 mg/kg in BALB/c mice. In the context of microbicide development, toxicity of GRFT at 2 mg/kg was determined after subcutaneous, intravaginal, and intraperitoneal administrations, respectively. Interestingly, GRFT caused no significant cell death, mitogenicity, activation, or cytokine release in mPBMCs, validating the usefulness of a mouse model. An excellent safety profile for GRFT was obtained in vivo: no overt changes were observed inanimal fitness, blood chemistry or CBC parameters. Following GRFT treatment, reversible splenomegaly was observed with activation of certain spleen B and T cells. However, spleen tissues were not pathologically altered by GRFT (either with a single high dose or chronic doses). Finally, no detectable toxicity was found after mucosal or systemic treatment with 2 mg/kg GRFT, which should be further developed as a microbicide for HIV prevention.

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PMID: 

Antimicrob Agents Chemother. 2020 May 21 ;64(6). Epub 2020 May 21. PMID: 32229493

Abstract Title: 

Rapid-Release Griffithsin Fibers for Dual Prevention of HSV-2 and HIV-1 Infections.

Abstract: 

The biologic griffithsin (GRFT) has recently emerged as a candidate to safely prevent sexually transmitted infections (STIs), including human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus 2 (HSV-2). However, to date, there are few delivery platforms that are available to effectively deliver biologics to the female reproductive tract (FRT). The goal of this work was to evaluate rapid-release polyethylene oxide (PEO), polyvinyl alcohol (PVA), and polyvinylpyrrolidone (PVP) fibers that incorporate GRFT in(HIV-1 and HSV-2) and(HSV-2) infection models. GRFT loading was determined via enzyme-linked immunosorbent assay (ELISA), and the bioactivity of GRFT fibers was assessed usingHIV-1 pseudovirus and HSV-2 plaque assays. Afterwards, the efficacy of GRFT fibers was assessed in a murine model of lethal HSV-2 infection. Finally, murine reproductive tracts and vaginal lavage samples were evaluated for histology and cytokine expression, 24 and 72 h after fiber administration, to determine safety. All rapid-release formulations achieved high levels of GRFT incorporation and were completely efficacious againstHIV-1 and HSV-2 infections. Importantly, all rapid-release GRFT fibers provided potent protection in a murine model of HSV-2 infection. Moreover, histology and cytokine levels, evaluated from collected murine reproductive tissues and vaginal lavage samples treated with blank fibers, showed no increased cytokine production or histological aberrations, demonstrating the preliminary safety of rapid-release GRFT fibers in vaginal tissue.

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PMID: 

Virology. 2012 Feb 20 ;423(2):175-86. Epub 2011 Dec 29. PMID: 22209231

Abstract Title: 

The lectins griffithsin, cyanovirin-N and scytovirin inhibit HIV-1 binding to the DC-SIGN receptor and transfer to CD4(+) cells.

Abstract: 

It is generally believed that during the sexual transmission of HIV-1, the glycan-specific DC-SIGN receptor binds the virus and mediates its transfer to CD4(+) cells. The lectins griffithsin (GRFT), cyanovirin-N (CV-N) and scytovirin (SVN) inhibit HIV-1 infection by binding to mannose-rich glycans on gp120. We measured the ability of these lectins to inhibit both the HIV-1 binding to DC-SIGN and the DC-SIGN-mediated HIV-1 infection of CD4(+) cells. While GRFT, CV-N and SVN were moderately inhibitory to DC-SIGN binding, they potently inhibited DC-SIGN-transfer of HIV-1. The introduction of the 234 glycosylation site abolished HIV-1 sensitivity to lectin inhibition of binding to DC-SIGN and virus transfer to susceptible cells. However, the addition of the 295 glycosylation site increased the inhibition of transfer. Our data suggest that GRFT, CV-N and SVN can block two important stages of the sexual transmission of HIV-1, DC-SIGN binding and transfer, supporting their further development as microbicides.

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PMID: 

Antimicrob Agents Chemother. 2015 Dec ;59(12):7290-8. Epub 2015 Sep 14. PMID: 26369967

Abstract Title: 

Griffithsin and Carrageenan Combination To Target Herpes Simplex Virus 2 and Human Papillomavirus.

Abstract: 

Extensive preclinical evaluation of griffithsin (GRFT) has identified this lectin to be a promising broad-spectrum microbicide. We set out to explore the antiviral properties of a GRFT and carrageenan (CG) combination product against herpes simplex virus 2 (HSV-2) and human papillomavirus (HPV) as well as determine the mechanism of action (MOA) of GRFT against both viruses. We performed the experiments in different cell lines, using time-of-addition and temperature dependence experiments to differentiate inhibition of viral attachment from entry and viral receptor internalization. Surface plasmon resonance (SPR) was used to assess GRFT binding to viral glycoproteins, and immunoprecipitation and immunohistochemistry were used to identify the specific glycoprotein involved. We determined the antiviral activity of GRFT against HSV-2 to be a 50% effective concentration (EC50) of 230 nM and provide the first evidence that GRFT has moderate anti-HPV activity (EC50 = 0.429 to 1.39μM). GRFT blocks the entry of HSV-2 and HPV into target cells but not the adsorption of HSV-2 and HPV onto target cells. The results of the SPR, immunoprecipitation, and immunohistochemistry analyses of HSV-2 combined suggest that GRFT may block viral entry by binding to HSV-2 glycoprotein D. Cell-based assays suggest anti-HPV activity through α6 integrin internalization. The GRFT-CG combination product but not GRFT or CG alone reduced HSV-2 vaginal infection in mice when given an hour before challenge (P = 0.0352). While GRFT significantly protected mice against vaginal HPV infection when dosed during and after HPV16 pseudovirus challenge (P

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PMID: 

Retrovirology. 2015 Jan 23 ;12:6. Epub 2015 Jan 23. PMID: 25613831

Abstract Title: 

Griffithsin tandemers: flexible and potent lectin inhibitors of the human immunodeficiency virus.

Abstract: 

BACKGROUND: The lectin griffithsin (GRFT) is a potent antiviral agent capable of prevention and treatment of infections caused by a number of enveloped viruses and is currently under development as an anti-HIV microbicide. In addition to its broad antiviral activity, GRFT is stable at high temperature and at a broad pH range, displays little toxicity and immunogenicity, and is amenable to large-scale manufacturing. Native GRFT is a domain-swapped homodimer that binds to viral envelope glycoproteins and has displayed mid-picomolar activity in cell-based anti-HIV assays. Previously, we have engineered and analyzed several monomeric forms of this lectin (mGRFT) with anti-HIV EC50 values ranging up to 323 nM. Based on our previous analysis of mGRFT, we hypothesized that the orientation and spacing of the carbohydrate binding domains GRFT were key to its antiviral activity.RESULTS: Here we present data on engineered tandem repeats of mGRFT (mGRFT tandemers) with antiviral activity at concentrations as low as one picomolar in whole-cell anti-HIV assays. mGRFT tandemers were analyzed thermodynamically, both individually and in complex with HIV-1 gp120. We also demonstrate by dynamic light scattering and cryo-electron microscopy that mGRFT tandemers do not aggregate HIV virions. This establishes that, although the intra-virion crosslinking of HIV envelope glycoproteins is likely integral to their activity, the antiviral activity of these lectins is not due to virus aggregation caused by inter-virion crosslinking.CONCLUSIONS: The engineered tandemer constructs of mGRFT may provide novel and powerful agents for prevention of infection by HIV and other enveloped viruses.

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PMID: 

Antimicrob Agents Chemother. 2011 Jan ;55(1):264-75. Epub 2010 Oct 18. PMID: 20956603

Abstract Title: 

Potent strategy to inhibit HIV-1 by binding both gp120 and gp41.

Abstract: 

The development of an anti-HIV microbicide is critical in the fight against the spread of HIV. It is shown here that the covalent linking of compounds that bind gp120 with compounds that bind gp41 can inhibit HIV entry even more potently than individual inhibitors or noncovalent combinations. The most striking example involves griffithsin, a potent HIV inhibitor that binds to the surface of HIV gp120. While griffithsin inhibits HIV Env-mediated fusion in a CCR5-tropic cell-cell fusion assay with a 50% inhibitory concentration (IC(50)) of 1.31± 0.87 nM and the gp41-binding peptide C37 shows an IC(50) of 18.2 ± 7.6 nM, the covalently linked combination of griffithsin with C37 (Griff37) has an IC(50) of 0.15 ± 0.05 nM, exhibiting a potency 8.7-fold greater than that of griffithsin alone. Similarly, in CXCR4-tropic cell-cell fusion assays, Griff37 is 5.2-fold more potent than griffithsin alone. In viral assays, both griffithsin and Griff37 inhibit HIV replication at midpicomolar levels, but the linked compound Griff37 is severalfold more potent than griffithsin alone against both CCR5- and CXCR4-tropic virus strains. Another example of this strategy is the covalently linked combination of peptide C37 with a variant of the gp120-binding peptide CD4M33 (L. Martin et al., Nat. Biotechnol. 21:71-76, 2003). Also, nuclear magnetic resonance (NMR) spectra for several of these compounds are shown, including, to our knowledge, the first published NMR spectrum for griffithsin.

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PMID: 

J Infect Dis. 2020 May 11 ;221(Supplement_4):S480-S492. PMID: 32037447

Abstract Title: 

Griffithsin Inhibits Nipah Virus Entry and Fusion and Can Protect Syrian Golden Hamsters From Lethal Nipah Virus Challenge.

Abstract: 

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that causes fatal encephalitis and respiratory disease in humans. There is currently no approved therapeutic for human use against NiV infection. Griffithsin (GRFT) is high-mannose oligosaccharide binding lectin that has shown in vivo broad-spectrum activity against viruses, including severe acute respiratory syndrome coronavirus, human immunodeficiency virus 1, hepatitis C virus, and Japanese encephalitis virus. In this study, we evaluated the in vitro antiviral activities of GRFT and its synthetic trimeric tandemer (3mG) against NiV and other viruses from 4 virus families. The 3mG had comparatively greater potency than GRFT against NiV due to its enhanced ability to block NiV glycoprotein-induced syncytia formation. Our initial in vivo prophylactic evaluation of an oxidation-resistant GRFT (Q-GRFT) showed significant protection against lethal NiV challenge in Syrian golden hamsters. Our results warrant further development of Q-GRFT and 3mG as potential NiV therapeutics.

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PMID: 

Arch Virol. 2013 Feb ;158(2):349-58. Epub 2012 Oct 7. PMID: 23053519

Abstract Title: 

Griffithsin inhibits Japanese encephalitis virus infection in vitro and in vivo.

Abstract: 

Griffithsin (GRFT) is a broad-spectrum antiviral protein that is effective against several glycosylated viruses. Here, we have evaluated the in vitro and in vivo antiviral activities of GRFT against Japanese encephalitis virus (JEV) infection. In vitro experiments showed that treatment of JEV with GRFT before inoculation of BHK-21 cells inhibited infection in a dose-dependent manner, with 99 % inhibition at 100 μg/ml and a 50 % inhibitory concentration (IC(50)) of 265 ng/ml (20 nM). Binding assays suggested that binding of GRFT to JEV virions inhibited JEV infection. In vivo experiment showed that GRFT (5 mg/kg) administered intraperitoneally before virus infection could completely prevent mortality in mice challenged intraperitoneally with a lethal dose of JEV. Our study also suggested that GRFT prevents JEV infection at the entry phase by targeting the virus. Collectively, our data demonstrate that GRFT is an antiviral agent with potential application in the development of therapeutics against JEV or other flavivirus infections.

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