To the Editor:
Vaginal microbicides to prevent heterosexual transmission of the human immunodeficiency virus type 1 (HIV-1) are being actively developed for clinical use, and these agents offer a practical and cost-effective approach to block HIV-1 transmission at a primary site of mucosal exposure.1 Strategies to enhance the natural mucosal defenses in women include vaginal application of compounds with acid-buffering capacities to sustain the normally low vaginal pH during and after intercourse, thereby counteracting the neutralizing effects of semen.2 Two such agents are currently in clinical testing: BufferGel (Reprotect LLC, Baltimore, MD)3,4 and ACIDFORM.5-8 Data from both animal models and human clinical trials indicate that these agents are safe and well tolerated and, when administered vaginally, can achieve acidification in the pH range from 4.4 to 5.2.3-10 When used as undiluted gels and mixed with semen in vitro, these agents demonstrate significant buffering capacities that translate well into practical application.5 The safety and tolerability of both BufferGel and ACIDFORM have been tested in Phase 1 clinical trials in the United States3 and Brazil,6-8 respectively, and published data are available from Phase 1 clinical trials of BufferGel in four international sites.4 In the latter case, daily vaginal application of BufferGel for 14 days achieved a median vaginal pH of 4.5 in women from India, Thailand, Malawi, and Zimbabwe.4
The efficacy of acid-buffering microbicides to prevent HIV-1 heterosexual transmission will depend in part on the ability of these agents to inactivate primary HIV-1 isolates, notably non-clade B strains that are prevalent in Asia and Sub-Saharan Africa. In vitro data from early studies suggested that HIV-1 is rapidly inactivated at acidic pH; however, these studies relied largely on the use of laboratory-adapted HIV-1 isolates11-13 and a limited number of patient isolates from northern Europe, which are presumably clade B.14 Little information is available on the acid sensitivity of primary, non-clade B HIV-1 isolates, including those of different co-receptor tropisms.
In the present study, we sought to characterize the sensitivity of primary non-clade B HIV-1 isolates to inactivation under mildly acidic conditions that include the natural pH range found in the vaginal environment of healthy women. Primary HIV-1 isolates from clades A, B, C, and E with tropism for CCR5 (R5), CXCR4 (X4), or R5/X4 were diluted 1:10 with media (DMEM supplemented with 10% FBS, antibiotics, and L-glutamine) at either neutral pH 7.8 or acidified to pH 3.8 to 4.5 by the addition of a 30% aqueous solution of L-(+)-lactic acid (Sigma, St. Louis, MO). The HIV-1 isolates were incubated for 5-30 minutes at 37°C and then rapidly neutralized by addition to TZM-bl indicator cells (Program for Topical Prevention of Conception and Disease, Rush University, licensed by Instead). HIV-1 infectivity was determined by measuring the levels of luciferase activity in TZM-bl cell lysates 48 hours after infection (Bright-Glo Luciferase Assay System, Promega, Madison, WI). No adverse effects on cell viability were observed for cells exposed to acid-treated/neutralized HIV-1 (data not shown). The relative levels of infectious HIV-1 remaining after exposure to acidic pH were calculated as a percentage of control cultures in which each HIV-1 isolate was exposed to media at neutral pH 7.8 for 5 to 30 minutes before adding to TZM-bl cells.
Consistent with previous reports, we found that a laboratory-adapted strain of HIV-1 (HIV-1NL-43) was rapidly inactivated at pH <4.5 (Fig. 1, upper left) with a significant loss of activity occurring within 5 minutes at pH 3.8 and 4.0. A similar pattern of inactivation was also observed for several primary HIV-1 isolates, including X4, R5, and dual-tropic strains (Fig. 1, HIV-1HC4, HIV-1CaseC7/86, HIV-192TH001). Of the eight primary HIV-1 isolates tested, all were rapidly inactivated after exposure to pH 3.8 for 30 minutes. The majority (6/8) also quickly lost infectivity after exposure to pH 4.0. However, we identified two isolates (R5 HIV-1JR-FL and R5 HIV-1DJ258) that retained significant levels of infectivity (65%-75%) after 5 minutes at pH 4.0 and were only gradually inactivated over 30 minutes. By comparison, all of the primary HIV-1 isolates retained infectivity after treatment at pH 4.5 with most retaining >50% infectivity for up to 30 minutes. Of interest, we identified three primary HIV-1 isolates (Fig. 1, bottom row, HIV-1CaseC1/85, HIV-1DJ259, HIV-1CM235) that showed enhanced levels of infectivity at pH 4.5 compared to neutral pH controls, and this enhanced activity was retained for up to 30 minutes. Each of these isolates utilizes CCR5 for infectivity, but they are of different genetic clades (C, B, and E, respectively).
Overall, our results demonstrate considerable variability among primary HIV-1 isolates to inactivation at acidic pH 4.0 and 4.5, and in some cases HIV-1 infectivity was actually enhanced by exposure to mildly acidic conditions that fall within the pH range achieved by acid-buffering microbicides after vaginal application. The acid-stability of primary HIV-1 isolates at pH 4.5 supports earlier findings using a limited number of primary HIV-1 isolates,14 and we now extend this result to include non-clade B viruses of different co-receptor phenotypes.
In vivo, the impact of mildly acidic pH on HIV-1 infectivity is balanced by myriad factors that influence the vaginal environment during and after sexual intercourse. Several substances with antiviral activity, including secretory leukocyte protease inhibitor (SLPI), have been identified in cervicovaginal secretions and are likely to play a role in innate vaginal defense against HIV-1.15 Moreover, semen itself, although transiently neutralizing the vaginal pH, may also have antiviral effects on HIV-1.16 Based on our data, it is unlikely that exposure to pH ≥4.5 is alone capable of significantly inactivating most primary strains of HIV-1 and may, in fact, enhance the infectivity of certain isolates, including CCR5-tropic, non-clade B viruses. Whether this translates into increased transmission of HIV-1 across the vaginal epithelium in unknown, but it may be of importance for the future development and application of vaginal acid-buffering microbicides that seek to block HIV-1 transmission at this mucosal site.
Ruth I. Connor, PhD
Department of Microbiology and Immunology Dartmouth Medical School Lebanon, NH
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