The potential of broadly neutralizing HIV antibodies to function as microbicides

Microbicides are products designed for vaginal or rectal use to prevent transmission of the human immunodeficiency virus (HIV). The first generation non-antiretroviral (non-ARV) microbicide candidates were intended to be a low-cost, female-controlled method of HIV prophylaxis because young women in the poorest regions of the world are disproportionately affected by HIV. However, these early microbicide candidates were not HIV specific and some disrupted the vaginal epithelium, increased immune activation in the female genital tract, or disturbed vaginal flora, while others simply did not work. Due to the poor clinical success of these first-generation candidates, there was a shift in focus to developing antiretroviral (ARV) compounds like tenofovir and dapivirine as microbicides. However, ARV-based microbicides may not prevent transmission of drug-resistant HIV. Moreover, not all persons may want to use an ARV-based product due to the potential of drug side-effects and the risk of developing drug-resistance if the product is used inappropriately. While there has been progress in developing a product for oral HIV pre-exposure prophylaxis (PrEP), there are still no commercially available topical microbicide products. Topical microbicides are desirable because they deliver active agents directly to the vaginal or rectal mucosa where HIV transmission occurs while avoiding systemic drug exposure. Hence non-ARV based microbicides are of great public health significance as a user-controlled tool for reducing the sexual transmission of HIV toward achieving the 2030 Sustainable Development Goal of ending AIDS and ensuring good health and well-being for all. Consequently, several years after the failure of the first generation of non-ARV vaginal gel microbicides, the lessons learned from these early trials have given rise to more rigorous preclinical evaluation protocols and novel formulation and delivery technologies for microbicides. This has resulted in renewed interest and new approaches to developing non-ARV microbicides. The new generation of non-ARV microbicide candidates being developed includes active biologics like broadly neutralizing monoclonal antibodies. This dissertation presents a pre-clinical evaluation of the potential of unformulated monoclonal HIV neutralizing antibodies to function as topical HIV microbicides in vitro and using human ex vivo models of rectal and vaginal mucosal transmission

Zidovudine (AZT) Monotherapy Selects for the A360V Mutation in the Connection Domain of HIV-1 Reverse Transcriptase

Background: We previously demonstrated in vitro that zidovudine (AZT) selects for A371V in the connection domain and Q509L in ribonuclease H (RNase H) domain of HIV-1 reverse transcriptase (RT) which, together with the thymidine analog mutations D67N, K70R and T215F, confer greater than 100-fold AZT resistance. The goal of the current study was to determine whether AZT monotherapy in HIV-1 infected patients also selects the A371V, Q509L or other mutations in the C-terminal domains of HIV-1 RT. Methodology/Principal Findings: Full-length RT sequences in plasma obtained pre- and post-therapy were compared in 23 participants who received AZT monotherapy from the AIDS Clinical Trials Group study 175. Five of the 23 participants reached a primary study endpoint. Mutations significantly associated with AZT monotherapy included K70R (p = 0.003) and T215Y (p = 0.013) in the polymerase domain of HIV-1 RT, and A360V (p = 0.041) in the connection domain of HIV-1 RT. HIV-1 drug susceptibility assays demonstrated that A360V, either alone or in combination with thymidine analog mutations, decreased AZT susceptibility in recombinant viruses containing participant-derived full-length RT sequences or site-directed mutant RT. Biochemical studies revealed that A360V enhances the AZT-monophosphate excision activity of purified RT by significantly decreasing the frequency of secondary RNase H cleavage events that reduce the RNA/DNA duplex length and promote template/primer dissociation. Conclusions: The A360V mutation in the connection domain of RT was selected in HIV-infected individuals that received AZT monotherapy and contributed to AZT resistance. © 2012 Brehm et al

Zidovudine (AZT) Monotherapy Selects for the A360V Mutation in the Connection Domain of HIV-1 Reverse Transcriptase. PLoS One 2012; 7:e31558. at Pennsylvania State U niversity on M arch 4, 2016 http://cid.oxfordjournals.org/ D ow nloaded from Ac ce pt ed

Abstract Background: We previously demonstrated in vitro that zidovudine (AZT) selects for A371V in the connection domain and Q509L in ribonuclease H (RNase H) domain of HIV-1 reverse transcriptase (RT) which, together with the thymidine analog mutations D67N, K70R and T215F, confer greater than 100-fold AZT resistance. The goal of the current study was to determine whether AZT monotherapy in HIV-1 infected patients also selects the A371V, Q509L or other mutations in the Cterminal domains of HIV-1 RT

Mutations selected by AZT monotherapy.

a<p>Two-sided McNemar's exact test between pre-therapy and AZT-experienced (N = 23 pairs). Not corrected for multiple comparisons.</p>b<p>TAMs listed in the IAS-USA 2010 drug resistance tables.</p

ATP-mediated AZT-MP excision activity and RNase H activity of wildtype, A360V, TAM-1 and TAM-1/A360V HIV-1 RT.

<p><b>A</b>) Isotherms of ATP-mediated AZT-MP excision reactions carried out by wildtype and mutant HIV-1 RT on a DNA/DNA T/P. Data are the mean ± standard deviation from at least three independent experiments. Reaction times were: wildtype and A360V = 10, 20, 30, 45, 60, 75, 90, 105 min; TAM-1 and TAM-1/A360V = 3, 7.5, 15, 25, 35, 45, 60, 75 min. <b>B</b>) Isotherms of ATP-mediated AZT-MP excision reactions carried out by wildtype and mutant HIV-1 RT on an RNA/DNA T/P. Data are the mean ± standard deviation from at least three independent experiments. Reaction times were: wildtype and A360V = 15, 30, 45, 60, 75, 90, 105, 120 min; TAM-1 and TAM-1/A360V = 3, 7.5, 15, 25, 35, 45, 60, 75 min. <b>C</b>) Representative autoradiogram of the RNase H cleavage activity of the wildtype and mutant HIV-1 RTs. Experiments were carried out as described in the <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031558#s4" target="_blank">Materials and Methods</a></i>. The reaction times were wildtype and A360V = 15, 30, 45, 60, 75, 90, 105, 120 min; TAM-1 and TAM-1/A360V = 3, 7.5, 15, 25, 35, 45, 60, 75 min. <b>D</b>) Isotherms for the accumulation of the −10 product formed by wildtype and mutant HIV-1 RT during AZT-MP excision.</p

AZT susceptibility of recombinant viruses containing participant-derived RT sequences.

a<p>Mean ± standard deviation from 3–11 independent experiments.</p>b<p>Fold-resistance calculated by dividing EC₅₀ of mutant virus by EC₅₀ of wildtype (WT).</p>c<p>Calculated using means of log₁₀ transformed EC₅₀ values and two-sided Student's <i>t</i> test.</p>d<p>Fold-resistance calculated by dividing EC₅₀ of 360 V virus by EC₅₀ of 360 A virus.</p>e<p>Calculated using means of log₁₀ transformed EC₅₀ values and two-sided Student's <i>t</i> test.</p>f<p>Wildtype is xxLAI 3D (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031558#s4" target="_blank">Methods</a>).</p

Pattern of emergence of resistance mutations among participants who selected A360V.

a<p>Sample obtained at earliest available time point after treatment initiation and which mutations were present compared with the pre-therapy sample.</p>b<p>Sample obtained at time point of last-on-therapy sample available. Participants 3 and 4 reached a study endpoint. Participants 6, 11, 29 and 31 did not reach a study end-point.</p>c<p>For participants 6 and 11, mutation A360V occurred in <25% of the viral population which is difficult to identify by population sequencing.</p

Mutations and polymorphisms associated with A360V.

a<p>Mutations/polymorphisms of the viral population in each participant sample compared to consensus subtype B RT (Los Alamos HIV Sequence Database);</p>b<p>TAMs highlighted in bold;</p>c<p>Mutations/polymorphisms of an individual participant-derived recombinant clone when compared to consensus subtype B RT (Los Alamos HIV Sequence Database).</p

AZT susceptibility of site-directed mutant HIV-1.

a<p>Wildtype (WT) is xxHIV-1_LAI.</p>b<p>Mean ± standard deviation from 4–6 independent experiments.</p>c<p>Average fold-resistance (Fold-R) of site-directed mutant EC₅₀ versus wildtype (WT).</p>d<p>Calculated using means of log₁₀ transformed EC₅₀ values and two-sided Student's <i>t</i> test.</p>e<p>Average Fold-R of 360 V versus A360 recombinant virus EC₅₀.</p>f<p>Calculated using means of log₁₀ transformed EC₅₀ values and two-sided Student's <i>t</i> test.</p