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Published in final edited form as: Virology. 2006 Sep 7;356(1-2):165–170. doi: 10.1016/j.virol.2006.08.006

Immunoglobulin A Antibodies against Internal HIV-1 Proteins Neutralize HIV-1 Replication inside Epithelial Cells

Alison Wright a, Huimin Yan a,1, Michael E Lamm a, Yung T Huang a,b,*
PMCID: PMC1797896  NIHMSID: NIHMS14204  PMID: 16956641

Abstract

We show that intraepithelial cell neutralization of HIV by IgA antibodies to internal viral proteins can occur during antibody transcytosis from the basolateral to the apical surface. Polarized epithelial cells expressing the polymeric immunoglobulin receptor (pIgR) were transfected with HIV proviral DNA, and IgA was added to the basolateral side. Transcytosing IgA antibodies against Gag and RT significantly inhibited HIV replication as assessed by infection of HeLa-CD4-LTR/β-Gal cells and direct p24 assay. Consistent with intracellular neutralization, colocalization of the internal virus proteins and their IgA antibodies was demonstrated by confocal microscopy. Thus, at least in the context of infections of polarized epithelia, antibody-mediated neutralization may not be restricted to viral surface antigens.

Keywords: Immunodeficiency diseases, Viral infection, Mucosa, Antibodies

Most heterosexual, homosexual and vertical infections with HIV occur through contact with a mucosal surface (Smith and Wahl, 2005), which acts as a gateway to the body. Suggested mechanisms of entry include viral transcytosis across epithelial cells or their tight junctions, infection of lymphoid cells through breaches in the epithelial lining, and infection of epithelial cells (Amerongen et al., 1991; Bomsel, 1997; Fantini et al., 1992; Fantini et al., 1991; Han et al., 2000; Hu et al., 2000; Lagaye et al., 2001; Moore et al., 2002; Moore et al., 2003; Qureshi et al., 1995; Spira et al., 1996).

The protective functions of the principal class of mucosal antibody, IgA, depend on pIgR-mediated endocytosis at the basolateral surface of epithelial cells and subsequent transcytosis. This process makes it possible for IgA to (a) block viral adherence or (b) bind virus in the lamina propria or in an epithelial cell and transport it into the lumen (Bomsel et al., 1998; Mazanec et al., 1995; Mazanec et al., 1992; Mazanec et al., 1993; Yan et al., 2002). Function (b) is referred to in general as “antigen excretion”, or in the particular case of viruses as “virus excretion” (Kaetzel et al., 1991; Robinson et al., 2001; Yan et al., 2002). On the other hand, we have used the term “intracellular neutralization” to describe antibody that inhibits virus production through an intracellular action (Mazanec et al., 1995; Mazanec et al., 1992; Mazanec et al., 1993; Yan et al., 2002). Such intraepithelial cell neutralization has been shown for IgA antibodies against Sendai virus, measles, influenza, rotavirus and HIV (Burns et al., 1996; Feng et al., 2002; Fujioka et al., 1998; Huang et al., 2005; Mazanec et al., 1992; Mazanec et al., 1993; Schwartz-Cornil et al., 2002; Yan et al., 2002).

Resistance to HIV has been correlated with the presence of specific mucosal IgA in uninfected sex workers and sexual partners of infected individuals in some but not all studies (Beyrer et al., 1999; Buchacz et al., 2001; Devito et al., 2002; Devito et al., 2000; Dorrell et al., 2000; Kaul et al., 2001; Kaul et al., 1999; Mazzoli et al., 1999; Mazzoli et al., 1997; Skurnick et al., 2002), and anti-HIV IgA able to neutralize T-cell line-adapted and primary HIV isolates has been collected from both infected humans, even though anti-HIV IgA responses tend not to be prominent (Burnett et al., 1994; Devito et al., 2000; Kozlowski et al., 1995; Mestecky et al., 2004; Moja et al., 2000; Wu et al., 2003), and mucosally immunized mice (Akagi et al., 2003; Bukawa et al., 1995; VanCott et al., 1998). Earlier we demonstrated that IgA antibodies against HIV envelope protein can mediate intracellular neutralization (Huang et al., 2005). The neutralization is thought to result from the fusion of transcytotic vesicles containing IgA antibody with vesicles containing envelope protein that have budded off the secretory pathway that is followed by proteins synthesized in the rough endoplasmic reticulum and destined for cell surface expression or export. In contrast to envelope proteins and the secretory pathway, the subcellular trafficking of internal viral proteins that are synthesized on free cytoplasmic ribosomes is less well understood. Here we show that the less variant internal HIV proteins Gag and RT can also be targets for antibody-mediated intracellular neutralization. The results have implications for the design of mucosal vaccines.

Murine IgA monoclonal antibodies (MAb) against RT and the structural protein p24 (Gag) were employed. The anti-Gag hybridoma, 183-H12-5C, which produces IgG1 (Chesebro et al., 1992), was obtained from the NIH AIDS Research and Reagent Program, and an IgA MAb switch variant was selected after repeated cycles of limiting dilution and spontaneous isotype switching (Boot et al., 1988). RT IgG and IgA hybridomas were produced by immunizing mice with RT protein (kindly provided by Stuart Le Grice, NIH). Production, purification, and characterization of MAbs were performed as described (Yan et al., 2002). IgA anti-gp120 was described in (Huang et al., 2005). The percent of oligomeric IgA, the form capable of binding to the pIgR, was 84% and 68% for the RT and Gag IgAs respectively.

Transport of RT and Gag IgA

The ability of the MAbs to transcytose a polarized epithelial membrane was tested with a monkey kidney cell line, Vero C1008, stably transfected to express human pIgR (Huang et al., 1999; Yan et al., 2002). Membrane polarization was tested by monitoring electrical resistance between apical and basal chambers. Transcytosis was assessed after adding 100 μg/mL MAb to the basolateral chamber. Apical supernatants were collected and analyzed by ELISA for Ig content. The amount of anti-RT IgA transported at 4, 8, and 12 h was 149±19, 433±25, and 557±37 ng, and for anti-Gag IgA was 73±13, 145±21, and 237±15 ng. In contrast, the maximum amount of IgG, which does not bind to pIgR, that reached the apical chamber at 12 h was 26±2 ng.

Intracellular HIV neutralization

The ability of the MAbs to neutralize HIV inside polarized epithelial cells was assayed as in (Huang et al., 2005). Briefly, polarized pIgR+ Vero cells were transfected with 2 μg of proviral HIV DNA (pKS242) with a 4 h incubation before the cells were washed and fresh medium added apically and either medium or MAb added to the basolateral chamber for 18 h. Then cells were washed and fresh media added. After a 12 h incubation the apical and basolateral media were collected. Monolayers were washed to remove extracellular virus, and cells were collected via scraping, freeze-thawed and centrifuged to create cell lysate. HeLa (CD4-LTR/β-Gal) cells expressing human CD4 and the HIV long terminal repeat fused to a LacZ reporter gene (Kimpton and Emerman, 1992) were infected with apical supernatant, basal medium, or cell lysate, and the blue infected cells were counted 48 h after infection (Huang et al., 2005).

Virus titers measured by the above blue cell assay were decreased 38% and 40% (p<0.005) in the apical supernatants by RT and Gag IgA and by 36% and 35% (p<0.005) in the cell lysates compared to controls without antibody (Table 1). Virus titers in the basal samples were too low to quantify (data not shown). The IgAs capable of mediating this neutralization were internalized by the pIgR since neither pIgR− cells with the same IgAs nor pIgR+ cells with anti-RT or anti-Gag IgG showed neutralization. Irrelevant IgA MAb (anti-measles virus hemagglutinin) did not neutralize, indicating the intraepithelial neutralization was immunologically specific (Table 1).

Table 1.

Intracellular neutralization of HIV by transcytosing IgA against internal HIV-1 proteins.a

Apical Supernatant
Cell Lysate
MAb (150μg/mL) Blue Cell # % Virus Reductionb Blue Cell # % Virus Reductionb
pIgR+ Cells
 Control 136 ± 16 1797 ± 194
 RT-IgA 84 ± 23 38c 1143 ± 200 36c
 RT-IgG 133 ± 20 3 1851 ± 197 0
 Gag-IgA 81 ± 18 40c 1176 ± 128 35c
 Gag-IgG 129 ± 17 5 1777 ± 164 1
 Irrelevant IgA 131 ± 27 4 1871 ± 357 0
pIgR− Cells
 Control 152 ± 12 1943 ± 134
 RT-IgA 155 ± 33 0 1872 ± 127 4
 Gag-IgA 159 ± 32 0 1933 ± 137 1
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a

Polarized monolayers of pIgR+ or pIgR− Vero C1008 cells were transfected with HIV proviral DNA for 4 h and then exposed to MAbs basolaterally for 18 h. HIV levels in apical supernatants and cell lysates 30 h post transfection were determined by infecting target HeLa cells (“blue cell” assay). Data are means from eight data points ± SEM pooled from four experiments.

b

Compared to the no antibody control.

c

Result significantly different (p<0.005) from the no antibody control according to a two-tailed t-test.

Concentration dependence of neutralization

Intracellular neutralization as a function of IgA antibody concentration is shown in Table 2. For both anti-RT and anti-Gag IgA, the reduction in virus titers decreased with lower antibody concentrations. This was true for both apical supernatants and cell lysates. A higher antibody concentration, 300 μg/mL, reduced virus production similarly to the 150 μg/mL concentration (data not shown).

Table 2.

IgA antibody concentration dependence of intracellular virus neutralization and comparison to conditioned media a

Apical Supernatant
Cell Lysate
IgA MAb (μg/mL) Blue Cell # % Virus Reductionb Blue Cell # % Virus Reductionb
Control 156 ± 26 1781 ± 123
Anti-RT (150) 96 ± 21 39 c 1075 ± 125 40c
Anti-RT (50) 115 ± 14 26c 1336 ± 252 25c
Anti-RT (20) 126 ± 34 19c 1674 ± 133 6c
Control 145 ± 21 1814 ± 76
Anti-Gag (150) 100 ± 16 31c 1330 ± 158 27c
Anti-Gag (50) 114 ± 16 21c 1489 ± 122 18c
Anti-Gag (20) 140 ± 23 4 1808 ± 202 1
Control 137 ± 15 1776 ± 43
Anti-RT (150) 82 ± 14 40c 1086 ± 99 39c
Anti-Gag (150) 80 ± 20 42c 1032 ± 138 42c
C.M.1- Anti-RTd 125 ± 33 9 1707 ± 114 4
C.M.1- Anti-GAGd 125 ± 20 9 1774 ± 124 2
C.M.2- Anti-RTe 1717 ± 159 3
C.M.2- Anti-GAGe 1774 ± 124 0
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a

Experiments as in Table 1 except that only pIgR+ epithelial cells were used. In some cases conditioned medium (C.M.) was added to the apical chamber or cell lysate without MAb having been added to the basolateral surface. Data are means from eight data points ± SEM pooled from four experiments.

b

Compared to no antibody control.

c

Result significantly different (p≤0.03) from the no antibody control.

d

C.M. 1 - apical supernatant with transcytosed IgA (see text).

e

C.M. 2 - IgA-transcytosing cell lysate (see text).

Confirmation that neutralization is intraepithelial

To verify that the neutralization observed was an intracellular event, two conditioned media (C.M.) were prepared (Huang et al., 2005). To test if neutralization could be due to transcytosed IgA in the apical compartment, C.M.1 was made from apical supernatant of uninfected monolayers exposed basolaterally to MAb. C.M.1 was then added apically to infected monolayers not exposed to antibody basolaterally for the final 12 h of incubation before collection as usual. The results in Table 2 show that the apical supernatant viral titers for C.M.1 of anti-RT and anti-Gag IgAs both had a 9% (p>0.14) decrease compared to the significant decrease of 40–42% (p<0.005) when anti-RT and anti-Gag IgAs were added basolaterally. Note that these MAbs against internal HIV proteins lack conventional neutralizing activity, even at 50 μg/ml (data not shown). C.M.1 also did not decrease viral titers in cell lysates (4% versus 39% for transcytosing anti-RT (p<0.005) and 2% versus 42% for transcytosing anti-Gag (p<0.005)). The finding that transcytosing IgA antibodies reduced virus titers in apical supernatants and cell lysates significantly more than previously transcytosed IgA antibodies in C.M.1 supports intraepithelial neutralization.

The other conditioned medium, C.M.2, was used to show that neutralization did not result from the freeze-thaw cycle releasing HIV and free antibody that subsequently complexed. C.M.2 was generated from uninfected monolayers exposed basolaterally to antibody, whose cells were then collected and lysed. C.M.2 was used as the collection medium for infected cell monolayers that were not exposed to MAb basolaterally. C.M.2 containing anti-RT and anti-Gag yielded virus reductions of 3% and 0% (Table 2) versus 39% and 42% for the transcytosing antibodies in the standard assay. These results further support the interpretation that the virus neutralization observed was due to IgA antibody binding viral antigen while the antibody was transversing the cell.

Our data do not directly address the intracellular site where transcytosing IgA antibody meets Gag and RT, both of which are synthesized on cytoplasmic ribosomes. Nevertheless, because both IgA and Gag utilize endosomes and multivesicular bodies in their cellular trafficking (Apodaca et al., 1994; Gibson et al., 1998; Nydegger et al., 2003; Ono and Freed, 2004; Spearman, 2006; von Schwedler et al., 2003) it seems reasonable to suggest a confluence of such vesicles as a likely point of intersection.

Effect on p24 synthesis

To assess more directly the effect of transcytosing IgA antibody on virus synthesis, p24 was measured by ELISA in apical supernatant, basal medium and cell lysate (Huang et al., 2005). Concentrations of p24 in apical and basal media and cell lysates were reduced by 11%, 18%, and 11% respectively by anti-RT IgA compared to the no antibody control (all p’s ≤ 0.01) (Table 3). For anti-Gag IgA the reductions were 10%, 19%, and 11% (all p’s<0.02). That p24 was decreased in all three compartments by both anti-Gag and anti-RT IgA antibody confirms that the reduced yield of virus observed was not a result of intracellular virus being diverted to the apical compartment.

Table 3.

Direct measurement of p24 concentrations.a

Apical Supernatant
Basal Medium
Cell Lysate
IgA MAb (150μg/mL) p24 (ng/mL) % Virus Reductionb p24 (ng/mL) % Virus Reductionb p24 (ng/mL) % Virus Reductionb
Control 7.0 ± 0.9 7.7 ± 0.9 29.9 ± 4.0
Anti-RT 6.2 ± 1.4 11c 6.3 ± 0.5 18c 26.7 ± 4.2 11c
Anti-Gag 6.3 ± 1.4 10c 6.2 ± 1.7 19c 26.7 ± 2.3 11c
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a

Experiments as in Table 1 except that p24 concentrations were measured. MAbs were added for 18 h to the basolateral surface of polarized monolayers of pIgR+ Vero C1008 cells that had been transfected with HIV proviral DNA for 4 h. HIV levels in apical supernatants, basolateral media and cell lysates 30 h post transfection were determined by p24 ELISA assay. Data are means from 11 data points ± SEM pooled from seven experiments.

b

Compared to the no antibody control.

c

Mean virus titer significantly less than control (p<0.02).

Intracellular colocalization of viral antigen and antibody

Two-color immunofluorescence confocal microscopy was used to visualize the interaction of transcytosing IgA antibody with viral protein inside transfected epithelial cells (Fig.1). Monolayers were treated as above, but after 18–24 h exposure to antibody basolaterally the monolayers were washed to remove free antibody, fixed, permeabilized, and stained (Huang et al., 2005). HIV proteins gp120, Gag, and RT were stained red with rhodamine, and IgA antibodies were stained green with fluorescein (Huang et al., 2005). The gp120 protein was used for comparison since it was previously demonstrated to colocalize with transcytosing IgA anti-gp120 (Huang et al., 2005). Merged images of IgA antibody and Gag or RT protein show colocalization in all three cell planes, apical, middle and basal. Irrelevant IgA did not show colocalization with the HIV proteins (RT protein shown).

Fig. 1.

Fig. 1

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Intracellular colocalization of IgA antibodies and HIV proteins. When transfected, 5–10% of the cells produce virus and the Gag and RT IgAs neutralize 30–40% of that virus. Apical (A), middle (M), and basal (B) horizontal sections through transfected cell monolayers were imaged. Each has a red (rhodamine) channel for virus protein, a green (fluorescein) channel for IgA antibody, and merged red and green channels. Colocalization of red and green signals creates a yellow-orange color. IgA antibodies and their HIV antigen showed colocalization in A, M and B sections whereas irrelevant IgA showed little to no colocalization. (Bar = 20μm)

Even though direct evidence for infection of mucosal epithelial cells under natural conditions in humans is lacking, because transmission of HIV usually occurs across mucosal surfaces, the observations that IgA antibodies against both envelope protein (Huang et al., 2005) and internal proteins (the present study) can inhibit viral replication inside epithelial cells may be relevant to vaccine strategies for HIV, as well as other viruses. In our in vitro system, anti-Gag and anti-RT IgA antibodies were less effective than anti-gp120 antibodies. However, there have been difficulties in developing effective vaccines with envelope protein antigens due to shielding by glycosylation and high mutability. Therefore, the ability of mucosal IgA antibodies to target the non-glycosylated and relatively invariant internal antigens of HIV may be worth considering in the design of mucosal vaccines.

More generally, studies of virus neutralization by antibodies have traditionally emphasized viral surface antigens. In contrast, the present and related studies argue that in certain contexts, such as mucosal epithelia, antibodies to the internal antigens of virus particles can play significant roles in host defense.

Acknowledgments

We thank the Confocal Microscopy Core in the Comprehensive Cancer Center of Case School of Medicine/University Hospitals of Cleveland (P30 CA43703-12) for use of their facility. This research was supported by NIH grants AI-36359 and CA-43703.

Footnotes

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