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"The introduction of a microbicide with 60% efficacy, introduced into 73 low-income countries, could avert 2.5 million HIV infections over three years in women, men and infants."
Topical Microbicide Assays for Inhibitors of Sexually Transmitted Virus Infections: The development of topical microbicides for use in the prevention of the sexual transmission of HIV has become a high priority of the NIH and World Health Organization (WHO). Our laboratories have been at the forefront of topical microbicide development over the past decade.
Compounds may be evaluated for inhibitory activity in CD4-dependent and CD4-independent virus transmission assays. Target cells expressing HIV-1 coreceptors with cell surface CD4 (GHOST X4/R5) or without cell surface CD4 (ME180, cervical epithelial cells) can be infected by cocultivation with HIV-infected lymphocytes. Endpoint quantification is performed by RT or p24 ELISA (efficacy) and XTT (toxicity).
Compounds may also be evaluated in the presence of mucin which simulates the mucopolysaccharides present in the vaginal and anal vaults and at various pH levels to further mimic the extracellular milieu required for the topical transmission of HIV. Simulated seminal and vaginal fluids are also used to mimic the environment in which the test compounds must function. Secondary evaluations include specific mechanism of action assays to identify compounds which are directly virucidal, or which inhibit virus attachment, fusion and reverse transcriptase. Additionally, compounds may be evaluated for toxicity to normal vaginal flora (i.e. effect on Lactobacillus sp.).
The development of topical microbicides is described in greater detail in the attached algorithm (see the Topical Microbicide Development Program).
The development of topical microbicides for use in the prevention of the sexual transmission of HIV has become a high priority of the National Institutes of Health (NIH) and World Health Organization (WHO). Our laboratories have been at the forefront of topical microbicide development for over the past decade. ImQuest’s microbicide development program utilizes a wide range of microbiological, biochemical and molecular technologies to assess the activity of agents against HIV, define the precise mechanism of action of potential microbicides using assays that closely mimic the microbicide environment, and analyze the potential toxicity of an inhibitor. |
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In Vitro Efficacy Screening Assays
Topical microbicides must be capable of inhibiting the replication of clinical strains of virus that are transmitted by both cell-free and cell-associated mechanisms targeting cells which may or may not express cell surface CD4, including T-cells, monocytes and dendritic cells. Antiviral screening assays may be performed in the presence of mucopolysaccharides, vaginal and seminal fluids to reflect the environment in which the compound must act. Representative assays include:
- Cell-free and cell-associated virus transmission assays
- CD4 dependent and CD4 independent transmission assays
- PBMC efficacy and toxicity assays (clinical subtype B, C or E)
- Efficacy in monocytes and dendritic cells
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In Vitro Efficacy Evaluation Against Clinically Relevant Strains of Virus and Cell Types
Active topical microbicides will be used throughout the world to suppress the transmission of HIV. Thus the topical microbicides must be capable of inhibiting virus strains that are found in geographical locales found throughout the world, and especially in the developing world. Inhibition of the transmission of drug resistant strains is an especially important factor that will be examined. Representative strains include:
- Various HIV-1 subtypes and HIV-2
- Drug resistant and multi-drug resistant virus strains
- CCR5, CXCR4 and dual-tropic virus strains
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Cellular Toxicity Evaluation and Toxicity to Vaginal Tissues
Effective microbicides need to be nontoxic to cells lining the vaginal and or rectal environments and should not induce any toxic effects on normal vaginal flora, which confer additional protection from the transmission and spread of infectious organisms. In vitro and ex vivo evaluations of the toxicity of candidate microbicides will confirm their relative safety compared to known toxic inhibitors such ad nonoxynol-9. |
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Mechanism of Topical Microbicide Action
Microbicidal compounds may inhibit early steps in virus replication (virus entry, cell-to-cell fusion or reverse transcription) or may directly inactivate virions and render them non-infectious. Mechanistic assays will provide confirmation of the means by which candidate microbicides effect HIV replication and include a variety of assays specific to the specific steps in HIV replication:
- Attachment inhibition
- Fusion inhibition
- Direct virus inactivation (virucidal activity)
- Reverse transcriptase inhibition
- CCR5 and CXCR4-tropic virus entry inhibition
- DC-SIGN co-receptor virus entry inhibition
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Range of Action Evaluations
The ideal microbicide will not only be active against HIV but will also be active against other sexually transmitted organisms that may be present at the time of infection and will also remain active in the presence of vaginal and seminal plasma as well as at pH conditions present in the vaginal and rectal vaults.
- Efficacy against range of HIV-1 subtypes
- Efficacy against resistant viruses
- Efficacy against HIV-2, SIV and SHIV
- Efficacy against other STIs
- Efficacy in presence of synthetic vaginal fluid and seminal plasma
- Efficacy at vaginal PH
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Combination assays with other potential topical microbicides
As with therapeutic HIV inhibitors it is probable that a combination microbicide product which is highly efficacious and nontoxic and which targets multiple steps in HIV replication will be a highly effective HIV infection prevention strategy. Combination assays will be performed in traditional PBMC-based assays as well as with our trademark microbicide-like combination assays. |
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Resistance Evaluations
Throughout the world, the frequency of transmission of drug-resistant virus is undoubtedly on the rise and likely exceeds 15%. Our standard microbicide evaluation assays have been adapted to assess the activity of a microbicide against viruses with resistance-engendering mutations. |
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Virus Transmission Inhibition Evaluation
One of the most critical and overlooked property of an efficacious microbicide is the innate ability to prevent the transmission of virus through cell-to-cell and cell-free mechanisms. ImQuest has developed a specialized means to sensitively evaluate transmission and define the concentration of a microbicide that is required to totally suppress virus transmission. |
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Cervical Explant and Other Ex Vivo Model
Active topical microbicides will be evaluated against appropriate clinical and CXCR4- and CCR5-tropic viruses using cervical explant and other ex vivo tissue culture models. |
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Activity in candidate gel formulations
The final microbicide product will most likely be in the form of a gel and the activity, toxicity and stability of the topical microbicide in the selected final formulation must be evaluated. |
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Animal models
We will work with preferred providers to assess toxicity (rabbit vaginal irritation, rabbit penile irritation) and efficacy (non-human primate model of vaginal and rectal infection) of both the unformulated and formulated product. |
