Plasma concentration of CD4-IgG2 in treated animals.

<p>Animals administered 200 mg of CD4-IgG2 showed plasma concentrations in the range of 500 to 1400 ng/ml at the time of challenge. No apparent correlation between plasma concentration and protection was observed. The CD4-IgG2 concentration at the time of challenge in animals administered 20 mg was 100 ng/ml for 1 animal and below the limit of detection (8 ng/ml) for 2 animals. Serum samples from the remaining 3 animals administered 20 mg were unavailable for this analysis.</p

Protection of CD4-IgG2-treated rhesus macaques in a high-dose SIVmac239 challenge experiment.

<p>To maintain serum concentrations, CD4-IgG2 (or control human polyclonal IgG) was administered subcutaneously over a two-week period by an ALZET osmotic pump. Animals were challenged intrarectally with a single high dose inoculum (3–5×10³ TCID₅₀) of SIVmac239 3-days after initiation of CD4-IgG2 administration. (<b>A</b>) Viral loads for animals treated with 200 mg of control polyclonal human IgG as a function of time following SIVmac239 challenge. All control animals became infected. (<b>B</b>) Viral loads for animals administered 20 mg CD4-IgG2 as a function of time following SIVmac239 challenge. Three out of 6 animals were fully protected and one infected animal showed delayed primary viremia. Due to a technical problem with the ALZET osmotic pump, one of the protected animals (98045) did not receive the full dose of 20 mg but this animal did not become infected. (<b>C</b>) Viral loads for animals administered 200 mg CD4-IgG2 as a function of time following SIVmac239 challenge. Five out of 7 animals were protected and showed no sign of infection at any time point. The minimum detection level was 125 SIV RNA copies/ml with a 95% confidence level. Open symbol indicates protected animal, closed symbol indicates infected animal.</p

Anti-human CD4 response in animals treated with CD4-IgG2.

<p>Animal sera were tested in a human CD4-specific ELISA to detect macaque antibody responses against CD4-IgG2. Serum samples were tested up to 23 days post-viral challenge and no responses were detected before day 15, indicating that the animal protection outcome was independent of a response against human CD4. Serum samples from 3 animals administered 20 mg CD4-IgG2 were unavailable for this analysis.</p

FcRn is responsible for pH/antibody-mediated enhanced transcytosis of HIV-1.

<p>(<b>A</b>) Knockdown of FcRn eliminates enhanced transcytosis. Transcytosis was performed with the wild-type and FcRn knockdown HEC-1A cells in parallel at pH 6.0 or 7.4 using HIV-1_US657 (average inoculum = 712,745 RNA copies) and mAb 2F5 or control mAb (Den3). <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003776#s2" target="_blank">Results</a> represent mean+SE of three independent experiments; <i>p</i> = 0.032 comparing wild type and knockdown cells using 2F5 at pH 6.0 (repeated-measures ANCOVA). (<b>B</b>) An Fc mutant of mAb b12 that increases FcRn binding (M428L) increases transcytosis of HIV-1_JRFL at pH 6.0, whereas a mutant that decreases FcRn binding (I253A) reduces transcytosis. Data represent the mean+SE of two independent experiments. (<b>C</b>)(<b>D</b>) IgG1, but not IgA1 or IgA2, enhances transcytosis at pH 6.0. mAb IgG1 b12 or its dimeric (dIgA2 b12) and monomeric (mIgA2 b12) class-switched versions and mAb IgG1 HGN194 or class switched variants dIgA1 HGN194 and dIgA2 HGN194 were tested for transcytosis using (<b>B</b>) HIV-1_JRFL Env-pseudotyped virus (average inoculum = 2,269,529 RNA copies) or (<b>C</b>) SHIV_1157ipEL-p (average inoculum = 682,724 RNA copies). Fm-6 is an anti-SARS coronavirus IgG1 used as a negative control. All mAbs were tested at 50 µg/ml. Data represent mean+SE of two independent assays for each virus. With either virus, IgG1 versions of the mAbs allow significantly more transcytosis at pH 6.0 than do IgA versions combined (<i>p</i> = 0.025 for HIV-1_JRFL pseudoviruses and <i>p</i> = 0.021 for SHIV_1157ipEL-p, Kruskal-Wallis test); there is no significant difference between IgG and IgA versions at pH 7.4 (<i>p</i>>0.05).</p

HIV-specific IgG and low pH enhance HIV-1 transcytosis.

<p>(<b>A</b>) Indicated concentrations of HIV-specific IgG (HIVIG) or control IgG (IVIG) and 2 ng of p24 (average of 3,814,910 RNA copies) of HIV-1_US712 were added to the apical surface of HEC-1A cells at pH 6.0 or 7.4. Subnatant fluid virus was quantified by RT-PCR and expressed as a percentage of inoculum applied to the apical surface of the HEC-1A cells. Data represent mean+SE of eight independent experiments (some antibody concentrations were tested less frequently); <i>p</i> = 3.6×10⁻⁸ comparing HIVIG at pH 6.0 with IVIG at pH 6.0; <i>p</i> = 3.4×10⁻⁶ comparing HIVIG at pH 6.0 with HIVIG at pH 7.4 (Kruskal-Wallis test). (<b>B</b>) Enhanced transcytosis occurs with multiple HIV-1 strains, including clade B R5 (HIV-1_US657), R5/X4 (HIV-1_HT593), and X4 (HIV-1_HT599) strains. Data represent mean+SE of three independent experiments for HIV-1_US657 (average inoculum = 1,096,491 RNA copies) and one experiment each for HIV-1_HT593 (inoculum = 2,450,255 RNA copies) and HIV-1_HT599 (inoculum = 1,477,802 RNA copies). (<b>C</b>) IgG enhances transcytosis of clade C transmitted/founder Env-pseudotyped virus. IgG from serum of clade C-infected or uninfected subjects was used at 50 µg/ml. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003776#s2" target="_blank">Results</a> are mean+SE of two independent experiments (average inoculum = 1,556,522 RNA copies) at pH 6.0 only; <i>p</i> = 0.008 comparing clade C and HIV-Neg IgG (Kruskal-Wallis test).</p

Enhancement of HIV-1 transcytosis occurs with low viral inocula¹.

1<p>Transcytosis carried out at pH 6.0 with 200 µg/ml of HIVIG or 100 µg/ml mAb 2F5; data are representative of three independent experiments.</p

Effect of antibodies on transcytosis and infectivity.

<p>Transcytosis (pH 6.0) of HIV-1_JRFL by (<b>A</b>) poorly neutralizing HIVIG and mAbs b6 and F240 and (<b>B</b>) neutralizing mAbs 4E10, 2F5, 2G12, VRC01 and b12. HIV-1_JRFL average inoculum = 3,990,745 RNA copies. Dotted lines represent mean+2 SD of % virus transcytosed with Den3 control mAb. There are significant differences between the Den3 control and 2F5 (<i>p</i> = 0.0002), b12 (<i>p</i> = 0.007), 2G12 (<i>p</i> = 0.006), 4E10 (<i>p</i> = 0.001), b6 (<i>p</i> = 0.008), F240 (<i>p</i> = 0.002), and VRC01 (<i>p</i> = 0.003) and between IVIG (not shown) and HIVIG (<i>p</i> = 0.033) (repeated-measures ANCOVA). (<b>C</b>) Infectivity of HIV-1_JRFL whose transcytosis was mediated by poorly neutralizing or (<b>D</b>) neutralizing antibodies. All antibodies resulted in transcytosed virus that was infectious (>mean+2 SDs of relative light units [RLUs] from uninfected TZM-bl cells [dotted line]); average infectious inoculum = 244,601 RLUs. Compared to Den3 control, less infectious virus was transcytosed with b12 (<i>p</i> = 3.4×10⁻¹¹), VRC01 (<i>p</i> = 6.9×10⁻¹⁰), and 2G12 (<i>p</i> = 3.6×10⁻⁴) (repeated-measures ANCOVA). Compared to Den3, there was more infectious virus with F240 (<i>p</i> = 0.036) and with b6 (<i>p</i> = 0.068). Compared to IVIG (not shown), HIVIG resulted in more infectious virus (<i>p</i> = 0.003). Data in <b>A</b>–<b>D</b> represent mean+SE of three or four independent experiments. (<b>E</b>) Ratio of % transcytosed:% infectious virus from data in (<b>A</b>) and (<b>B</b>) using 50 µg/ml of antibody. Numbers over bars represent IC₅₀ against HIV-1_JRFL.</p

FcRn expression in human genital tissues, detected by immunohistochemistry.

<p>FcRn expression (red stain) in columnar epithelia lining (<b>A</b>) the penile urethra (representative of 16 donors), (<b>B</b>) endocervix (representative of five donors), or (<b>C</b>) vagina (representative of 12 donors). Tissues were also stained with negative control (non-specific) IgG and processed through the same immunohistochemistry procedure: (<b>D</b>) penile urethra, (<b>E</b>) endocervix, and (<b>F</b>) vaginal epithelia. Epithelia (EP) and lamina propria (LP) are labeled. Staining was performed on paraffin-embedded specimens.</p