Preclinical safety assessment of Griffithsin-based vaginal microbicides.

Griffithsin (GRFT) is a protein derived from the red alga Griffithsia sp. off the coast of New Zealand. It binds and inactivates number of enveloped viruses including HIV-1. For a product to be developed as a microbicide, it must be safe and effective after repeated use. For initial in vivo safety testing we exposed mice repeatedly to various vaginal products and found that increased concentration of mouse serum albumin (MSA) in the vaginal lumen is predictive of epithelial damage. Repeated intravaginal administration of GRFT did not result in increased amounts of MSA, but the known epithelial disrupters nonoxynol-9 and benzylkonium chloride caused increased concentrations of MSA that were detectable by ELISA and western blot. We also evaluated the immunogenicity of GRFT following repeated exposure and found that intravaginal administration resulted in higher serum IgG antibody titers, but not local IgA or IgG levels, than did subcutaneous administration. The consequences of the systemic immune response are unknown and require further investigation

Implementation of the Louisville COVID-19 Surveillance Protocol: Experiences from the University of Louisville Center of Excellence for Research in Infectious Diseases [CERID]

The lack of available testing for SARS-CoV-2 has been one of the primary challenges in the development and implementation of a comprehensive approach to infection prevention and transmission in the United States (US). In response to the need for increased testing capacities and capabilities, the University of Louisville (UofL) Division of Infectious Diseases, Center of Excellence for Research in Infectious Diseases (CERID) initiated the Louisville Coronavirus Surveillance Program, a comprehensive approach to surveillance and testing of patients and healthcare workers. The first specimens were accepted on March 12, 2020 and parallel testing was done using a high-capacity testing process and the Division of Infectious Diseases CLIA-certified laboratory to ensure concordant results. Steps in the testing process began with validation of the testing methods and included database development, acceptance of specimens, tracking and cataloging the specimens, testing, and reporting of results. Quality metrics were developed and used to prevent error and facilitate rapid reporting. Between March 12, 2020 and April 30, 2020, more than 5500 tests were performed identifying more than 850 patients and healthcare workers infected with COVID-19 in the Louisville, Kentucky area. Although the process used high-capacity robotics for testing procedures, the methods described here are applicable to settings employing a variety of laboratory testing methods

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

Griffithsin (GRFT), a lectin from Griffithsia species, 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 in animal 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

Occluding the Mannose Moieties on Human Immunodeficiency Virus Type 1 gp120 with Griffithsin Improves the Antibody Responses to Both Proteins in Mice

To assess the influence of mannosylated glycans on the immunogenicity of human immunodeficiency virus type 1 (HIV-1) Env proteins, we immunized mice with monomeric gp120 in the presence and absence of the mannose-binding protein, griffithsin (GRFT). For comparison, other groups of mice received the nonglycosylated HIV-1 Gag protein, with and without GRFT. Coimmunization with GRFT increased the anti-gp120 IgG reactivity significantly, but had no effect on the anti-Gag response. We also investigated the IgG response to GRFT and found that gp120, but not Gag, enhanced its immunogenicity. For both proteins, IgG1 antibodies dominated the IgG response, with IgG2b as the next most prevalent subclass. We conclude that gp120-GRFT complexes are more immunogenic than the free proteins, for both components, and that occluding the mannose moieties on monomeric gp120 can improve the humoral immune response to this protei