The Journal of Infectious Diseases BRIEF REPORT

Antibodies and Acidic Environment Do Not Enhance HIV-1 Transcytosis Oscar A. Gonzaleza and Manish Sagar Department of Medicine, Boston University School of Medicine, Massachusetts

A limited number of human immunodeficiency virus type 1 (HIV-1) variants initially infect HIV-1–naive individuals. Recent studies imply that this may occur because generally inefficient transcytosis across intact mucosal surfaces could be enhanced for specific viruses with bound antibodies and in the presence of acidic pH. We found that transcytosis of both cell-free and cell-associated viruses with diverse envelopes was significantly decreased in the presence of either antibodies or plasma from chronically infected transmitting partners regardless of pH. Transmitted variants also did not have greater transmigration as compared to chronic-infection strains. Enhanced translocation capacity is unlikely to influence which HIV-1 variant establishes infection. Keywords. transcytosis; mucosal transmission; HIV-1 envelope; selection at transmission.

The majority of human immunodeficiency virus type 1 (HIV-1) infections in the world are acquired across mucosal surfaces, and it remains unclear how HIV-1 bypasses an intact mucosa to access the deeper-lying primary target cells. Studies have suggested that migration from the apical to the basolateral surface of the exposed mucosa may occur through transcytosis, rather than through productive infection of epithelium-based cells [1]. During transcytosis, HIV-1 is presumed to enter an endosomelike structure within the epithelial cells in an envelope-dependent fashion. Recent studies show that the presence of antibodies and acidic pH at the apical surface enhances transcytosis across polarized epithelial cell monolayers [2, 3]. These reports contend that the vaginal acidic pH, which may fluctuate from around 4.5 to 6.0 before and after sexual activity because of semen alkalinity and the presence of lactobacilli [4], promotes attachment between an antibody-bound virus and the Fc neonatal receptor (FcRn) on the apical surface of some genital columnar cell. This immune complex is subsequently internalized and transported to the basolateral surface, where the virus is released in the presence of

Received 30 June 2016; accepted 30 July 2016; published online 4 August 2016. a Present address: Karyopharm Therapeutics, Needham, MA 02459. Correspondence: M. Sagar, Boston University, 650 Albany St, Rm 647, Boston, MA 02118 ([email protected]). The Journal of Infectious Diseases® 2016;214:1221–4 © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail [email protected]. DOI: 10.1093/infdis/jiw354

neutral pH. Importantly, this model implies that antibodybound viruses as opposed to HIV-1 variants with no or minimal amounts of bound antibody are potentially favored for transcytosis across mucosal surfaces. HIV-1–naive, exposed individuals are infected with a limited number of variants, even though the transmitting partner may harbor a diverse swarm [5]. Reasons for this selection bottleneck during mucosal HIV-1 acquisition remain unclear. Some but not all studies have suggested that the infecting strains are more sensitive to neutralization, compared with the nontransmitted variants [6]. We and others have also shown that the infecting strains often have ancestral properties, such as shorter and less glycosylated envelopes, which enhance neutralization susceptibility and antibody binding [6, 7]. Thus, it is possible that the relatively neutralization-sensitive infecting strains with ancestral characteristics have greater transcytosis capacity because they are more likely to have attached antibodies as compared to the majority of the quasispecies circulating in the chronically infected host. In this study, we examined whether variants from newly infected subjects demonstrated greater pH and antibody-dependent transcytosis across in vitro cellular monolayers, compared with strains circulating in the transmitting partner. METHODS Viruses

A summary of the strains used in this study is available in the Supplementary Materials. Virus stocks were prepared by transfecting HEK293T cells [7, 8]. Magnetic bead–isolated CD4+ T cells (Miltenyi Biotech) were activated with phytohemagglutinin and interleukin 2 and then exposed to infectious virus in the presence of diethylaminoethyl dextran [8]. After 7 days, infected cells were washed 4 times with phosphate-buffered saline and stored in aliquots of freezing medium. The p24gag antigen content of the cell-free and infected-cell virus stocks was estimated using a commercial enzyme-linked immunosorbent assay (Perkin Elmer). Use of samples from all subjects was with informed consent and approved by institutional review boards [8]. Antibodies and Plasma

Monoclonal immunoglobulin G1 (IgG1) antibody, b12, and HIVIG (IgG derived from pooled plasma from HIV-infected individuals) was obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. HIV-1–negative IgG (IVIG) was derived from human plasma (Lonza) by the IgG spin purification kit (Thermo Scientific). Plasma from transmitting partners was heat inactivated at 56°C for 1 hour. HIV-1– specific antibodies of other isotypes were not readily available, although plasma samples presumably contain other isotypes, such as immunoglobulin M and immunoglobulin A. BRIEF REPORT



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Transcytosis Assay

Approximately 1.5 × 105 previously confirmed FcRn-expressing human endometrial carcinoma (HEC-1A) cells (ATCC) were seeded onto 0.4-µm polyethylene terephthalate membrane hanging transwell inserts (Millipore) in a volume of 400 µL of complete McCoy’s 5A medium (supplemented with bovine 2.5 mM L-glutamine [Gibco], 1% HEPES [Corning], 2% Penstrep [Cellgro Mediatech], and 10% fetal bovine serum [Atlas Biologicals]) [2]. Transwell chambers were placed in a 24-well dish with 900 µL of medium on the basolateral surface, and they were incubated at 37°C in 5% CO2 for 6–10 days. Medium was changed every 2–3 days. Transepithelial electrical resistance (TEER) was measured using EVOM Volt/Ohm (World Precision Instruments). After TEER measurement reached ≥300 Ω/cm2 (120 Ω.cm2), fresh medium was added to both the apical and basal side of the transwell. Different virus amounts and antibody concentrations were incubated at 37°C for up to 1 hour. Subsequently, the pH in the virus mixtures was changed by adding drops of 1 M HCL or 1 M NaOH, and the final pH was confirmed by a pH strips. All apical medium was removed

from the transwell, and the virus/antibody cocktail was added to the apical layer. After 18 hours of incubation, the basal medium was harvested and assessed for p24gag antigen. The pH of the apical medium and TEER was tested after incubation to confirm that these parameters remained relatively stable over time. Statistical Analysis

Percent transcytosis was estimated as the level of p24gag present in the basolateral compartment relative to the amount in the apical side. All viruses and conditions were analyzed in duplicate and repeated a minimum of 3 times. Differences between >2 groups and between 2 individual groups were examined by 1-way analysis of variance (ANOVA) or an unpaired t test. All P values are based on 2-sided tests. RESULTS

Before examining whether viruses present in a newly infected individual are preferentially transcytosed in the presence of antibodies present in the transmitting partner, experiments were done to confirm previous reports that HIV-1 envelope–specific

Figure 1. Antibodies inhibit transcytosis at acidic pH. A, Approximately 3 ng of p24gag NL4-3 was added to the apical surface in the presence of medium alone, 100 µg/mL immunoglobulin G (IgG) derived from pooled plasma from human immunodeficiency virus–infected individuals, 100 µg/mL HIV-1–negative IgG, or 50 µg/mL b12. After incubation, mixture pH was changed to either 6.0 (black bars) or 7.4 (white bars) by adding HCL or NaOH, respectively. Percentage transcytosis was estimated by calculating the ratio of p24gag recovered from the basolateral compartment to the amount applied to the apical surface. Each condition was examined in duplicate in a minimum of 5 independent experiments. B, The percentage of p24gag recovered from transwells with approximately 10 ng of p24gag NL4-3 (black bars) or Bal (white bar) infected CD4+ T cells added to the apical surface in the presence of medium alone or 100 µg/mL HIVIG at pH 6.0. Star above bars indicate that the percentage transcytosis was significantly different than 0% (P < .05 by the 1-sample t test). Each condition was examined in duplicate in 4 independent experiments. C and D, Transcytosis was examined for viruses with envelopes isolated from 2 couples, H-F (C) and SR-5 (D). The viruses contained envelopes from either the individual sampled before HIV-1 seroconversion (recipient/transmitted-founder [T-F]) or the corresponding transmitting sex partner (donor/chronic). Transcytosis of around 3 ng of p24gag was examined at either pH 6.0 or 7.4 in the presence or absence of a 1:100 dilution of the transmitting partner’s heat-inactivated plasma. Each condition was examined in duplicate in a minimum of 3 independent experiments. In each graph, bars show the average of all experiments and standard error of the mean. *P < .05.

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antibodies enhanced virus migration across a cellular monolayer in the presence of acidic pH [2, 3]. NL4-3 transcytosis was not enhanced in the presence of HIV-1–specific polyclonal (HIVIG) or monoclonal (b12) antibody regardless of the pH of the medium (Figure 1A). The 100 µg/mL HIVIG and 50 µg/mL b12 bound NL4-3 because the mixture of virus and antibody as compared to virus in medium alone produced around 90% less luciferase signal from exposed TZM-bl reporter cell line (data not shown). NL4-3 transcytosis was also not significantly different in the presence of nonspecific antibodies (IVIG). In aggregate, none of the conditions yielded significantly different level of transcytosis (P = .88 by 1-way ANOVA). NL4-3 transcytosis in the presence of 100 µg/mL HIVIG was also not greater, compared with medium alone, when the pH of the apical compartment was reduced to 4.0 (data not shown). Thus, in contrast to previous reports [2, 3], we found that antibodies did not enhance NL4-3 transcytosis across a monolayer in the presence of acidic pH. Transmigration of cell-associated virus has also been shown to be significantly inhibited in the presence of diverse polyclonal antibodies at a neutral pH [9, 10]. No report has examined whether cell-associated transcytosis is enhanced in the presence of HIV-1–specific antibodies at acidic pH. When the HEC-1A monolayers were exposed to infected CD4+ T cells in medium alone at a pH of 6.0, a median of 1.3% (range, 1.1%–1.3%) and 0.6% (range, 0.5%–0.7%) of NL4-3 and Bal initial inoculum, respectively, was recovered in the basolateral compartment. In the presence of HIVIG at pH 6.0, no virus was recovered from basolateral medium for the 2 different cell-associated virus strains (0% transcytosis). The cell-associated percentage transcytosis was significantly >0% (P < .05 by the 1-sample t test for the difference from the hypothetical mean) for both viruses in the absence of antibodies at acidic pH (Figure 1B). Thus, polyclonal antibodies inhibit cell-associated HIV-1 transcytosis at both neutral and acidic pH. While previous studies have reported that antibodies in the presence of acidic pH enhanced transcytosis of transmittedfounder (T-F) strains [2, 3], these investigations have not examined the migration of the infecting variants in the presence of antibodies present in the transmitting partner. Chronically infected individuals harbor polyclonal anti-HIV antibodies that are often especially potent against the strains transmitted to their sex partners, compared with the majority of the circulating variants [6]. Thus, the infecting strains may be favored for transcytosis across cellular membranes in the presence of antibodies present in the transmitting partner. Recombinant viruses were generated that harbored envelopes isolated either from the acutely infected individual identified before seroconversion or from the corresponding transmitting partner [8]. Recombinant virus transcytosis was examined in the absence or presence of heat-inactivated plasma from the transmitting partner at acidic and neutral pH. Among both recipient-donor pairs (H-F and

Figure 2. The level of transcytosis is not different among infecting strains as compared to chronic-infection strains. Approximately 3 ng of p24gag of transmitted-founder (T-F) strains (white bars) and chronic-infection strains (black bars) was added to apical surface at pH 7.4, and the percentage transcytosis was estimated as the ratio of p24gag recovered from the basolateral compartment to the amount applied to the apical surface. The different infectious molecular clones are labeled on the x-axis. Each strain was examined in duplicate in a minimum of 4 independent experiments. Bars show the average of all experiments and the standard error of the mean.

SR-5), transcytosis was significantly lower (P < .05 by the unpaired t test) in the presence as compared to the absence of donor plasma for the viruses with the recipient envelopes (Figure 1C and 1D). Transmigration for the variants with the donor envelope was also lower in the presence as opposed to the absence of donor plasma for both individuals, although the difference was only statistically significant for H-F (P < .05 by the unpaired t test). In addition, the level of transmigration was not substantially different at acidic pH, compared with neutral pH, in the presence of heat-inactivated plasma. The pH of the apical mixture was not significantly different before and after incubation, suggesting that the addition of plasma did not alter acidity over time. In aggregate, donor plasma containing anti-HIV antibodies generated over the course of infection diminished transcytosis of HIV-1 variants containing infecting strain envelopes. Even though full-length infectious molecular clone T-F strains have been shown to have unique characteristics, such as greater envelope expression [11], no study has examined whether T-F strains as compared to chronic-infection strains possess greater transmigration capacity. Transcytosis was similar between T-F variants and unrelated isolates from the chronic stage of infection (Figure 2). At a neutral pH and in the absence of antibodies, the percentage of virus recovered from the basolateral compartment relative to the initial inoculum was not different for the T-F strains (n = 9; median, 0.95%; range, 0.52%–1.70%) as compared to chronic-infection strains (n = 5; median, 0.68%; range, 0.32%–1.32%; P = .12 by the Wilcoxon rank sum test). In aggregate, our data suggest that the infecting BRIEF REPORT



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strains do not have greater transcytosis capacity, compared with chronic-infection strains. DISCUSSION

In this study, we showed that acidic pH does not promote the migration of cell-free and cell-associated virus across an FcRnexpressing columnar monolayer. In contrast to recent investigations [2, 3], we showed that polyclonal antibodies, such as HIVIG, decrease transcytosis regardless of the pH. Reasons for the different results in our studies from these previously published findings remain unclear because the in vitro methods, reagents, and conditions were often similar. While acidic conditions are often present in the vagina, the usual pH is unknown at other potential transmission sites, such as the endocervix, anus, or intestinal cavity, which are presumably more susceptible to transcytosis because of a columnar as opposed to a stratified squamous epithelium. Furthermore, bacterial vaginosis is associated with an enhanced risk of HIV-1 acquisition, rather than a decreased risk, even though the vaginal environment becomes more basic during this process [12]. In addition, in vitro, lactobacilli-generated lactic acid decreases HIV-1 infectiousness, independent of transcytosis [13]. In aggregate, these caveats and our in vitro studies suggest that acidity and antibodies are unlikely to promote FcRn-mediated HIV-1 transmigration and subsequent acquisition. In this study, we also show that the unique infecting HIV-1 strains do not demonstrate greater transcytosis capacity, compared with variants isolated either during the chronic stage of infection or from the transmitting partner. Secretions from HIV-1-infected transmitting partners contain antibodies that potentially preferentially bind variants with features overrepresented among the T-F strains, such as shorter and less glycosylated envelopes [6, 7, 14, 15]. Our results also show that recombinant viruses with envelopes from the infecting strain as compared to nontransmitted envelopes do not translocate more efficiently across cellular monolayers in the presence of antibodies present in the transmitting partner’s plasma, regardless of pH. Most contemporaneous antibodies are unable to neutralize circulating strains [5]. A larger set of infectious viruses isolated from transmitting partners could more definitively distinguish whether the infecting strains as compared to the majority of quasispecies present in the transmitting partner have enhanced transcytosis capacity especially in the presence of plasma from the chronically infected transmitter. Regardless, our experiments confirm previous studies [9, 10] and provide

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further evidence that polyclonal binding but not necessarily neutralizing antibodies present in chronically infected individuals inhibit transcytosis across cellular monolayers even in acidic conditions. Supplementary Data Supplementary materials are available at http://jid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author. Notes Acknowledgment. We thank Deborah Anderson for reviews and suggestions. Financial support. This work was supported by the National Institutes of Health (grant AI122209 to M. S.). Potential conflicts of interest. Both authors: No reported conflicts. Both authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. Bomsel M. Transcytosis of infectious human immunodeficiency virus across a tight human epithelial cell line barrier. Nat Med 1997; 3:42–7. 2. Gupta S, Gach JS, Becerra JC, et al. The Neonatal Fc receptor (FcRn) enhances human immunodeficiency virus type 1 (HIV-1) transcytosis across epithelial cells. PLoS Pathog 2013; 9:e1003776. 3. Gupta S, Pegu P, Venzon DJ, et al. Enhanced in vitro transcytosis of simian immunodeficiency virus mediated by vaccine-induced antibody predicts transmitted/founder strain number after rectal challenge. J Infect Dis 2015; 211:45–52. 4. Linhares IM, Summers PR, Larsen B, Giraldo PC, Witkin SS. Contemporary perspectives on vaginal pH and lactobacilli. Am J Obstet Gynecol 2011; 204:120 e1–5. 5. Sagar M. HIV-1 transmission biology: selection and characteristics of infecting viruses. J Infect Dis 2010; 202(suppl 2):S289–96. 6. Derdeyn CA, Decker JM, Bibollet-Ruche F, et al. Envelope-constrained neutralization-sensitive HIV-1 after heterosexual transmission. Science 2004; 303:2019–22. 7. Sagar M, Wu X, Lee S, Overbaugh J. HIV-1 V1-V2 envelope loop sequences expand and add glycosylation sites over the course of infection and these modifications affect antibody neutralization sensitivity. J Virol 2006; 80:9586–98. 8. Pena-Cruz V, Etemad B, Chatziandreou N, et al. HIV-1 envelope replication and α4β7 utilization among newly infected subjects and their corresponding heterosexual partners. Retrovirology 2013; 10:162. 9. Hocini H, Bomsel M. Infectious human immunodeficiency virus can rapidly penetrate a tight human epithelial barrier by transcytosis in a process impaired by mucosal immunoglobulins. J Infect Dis 1999; 179(suppl 3):S448–53. 10. Bomsel M, Heyman M, Hocini H, et al. Intracellular neutralization of HIV transcytosis across tight epithelial barriers by anti-HIV envelope protein dIgA or IgM. Immunity 1998; 9:277–87. 11. Parrish NF, Gao F, Li H, et al. Phenotypic properties of transmitted founder HIV1. Proc Natl Acad Sci U S A 2013; 110:6626–33. 12. Atashili J, Poole C, Ndumbe PM, Adimora AA, Smith JS. Bacterial vaginosis and HIV acquisition: a meta-analysis of published studies. Aids 2008; 22:1493–501. 13. Aldunate M, Tyssen D, Johnson A, et al. Vaginal concentrations of lactic acid potently inactivate HIV. J Antimicrob Chemother 2013; 68:2015–25. 14. Mkhize NN, Durgiah R, Ashley V, et al. Broadly neutralizing antibody specificities detected in the genital tract of HIV-1 infected women. AIDS 2016; 30:1005–14. 15. Raux M, Finkielsztejn L, Salmon-Ceron D, et al. IgG subclass distribution in serum and various mucosal fluids of HIV type 1-infected subjects. AIDS Res Hum Retroviruses 2000; 16:583–94.

Antibodies and Acidic Environment Do Not Enhance HIV-1 Transcytosis.

A limited number of human immunodeficiency virus type 1 (HIV-1) variants initially infect HIV-1-naive individuals. Recent studies imply that this may ...
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