Lymphatic Dissemination of Simian Immunodeficiency Virus after Penile Inoculation

Abstract

The human immunodeficiency virus (HIV) is primarily transmitted by heterosexual contact, and approximately equal numbers of men and women worldwide are infected with the virus. Understanding the biology of HIV acquisition and dissemination in men exposed to the virus by insertive penile intercourse is likely to help with the rational design of vaccines that can limit or prevent HIV transmission. To characterize the target cells and dissemination pathways involved in establishing systemic simian immunodeficiency virus (SIV) infection, we necropsied male rhesus macaques at 1, 3, 7, and 14 days after penile SIV inoculation and quantified the levels of unspliced SIV RNA and spliced SIV RNA in tissue lysates and the number of SIV RNA-positive cells in tissue sections. We found that penile (glans, foreskin, coronal sulcus) T cells and, to a lesser extent, macrophages and dendritic cells are primary targets of infection and that SIV rapidly reaches the regional lymph nodes. At 7 days after inoculation, SIV had disseminated to the blood, systemic lymph nodes, and mucosal lymphoid tissues. Further, at 7 days postinoculation (p.i.), spliced SIV RNA levels were the highest in the genital lymph nodes, indicating that this is the site where the infection is initially amplified. By 14 days p.i., spliced SIV RNA levels were high in all tissues, but they were the highest in the gastrointestinal tract, indicating that the primary site of virus replication had shifted from the genital lymph nodes to the gut. The stepwise pattern of virus replication and dissemination described here suggests that vaccine-elicited immune responses in the genital lymph nodes could help prevent infection after penile SIV challenge.

Importance: To be the most effective, vaccines should produce antiviral immune responses in the anatomic sites of virus replication. Thus, understanding the path taken by HIV from the mucosal surfaces, which are the site of virus exposure, to the deeper tissues where the virus replicates will provide insight into where AIDS vaccines should produce immunity to be the most effective. In this study, we determined that, by day 7 after penile inoculation, SIV has moved first to the inguinal lymph nodes and replicates to high levels. Although the virus is widely disseminated to other tissues by day 7, replication is largely limited to the inguinal lymph nodes. The step-by-step movement of SIV from penile mucosal surfaces to the draining lymph nodes may allow an HIV vaccine that produces immunity in these lymph nodes to block HIV from establishing an infection in an exposed person.

FIG 1

SIV RNA levels in plasma of rhesus macaques after penile SIVmac251 inoculation. Total vRNA levels in plasma were determined by RT-PCR. The last blood sample was collected at necropsy. The animal number associated with each symbol is indicated.

FIG 2

Unspliced and spliced vRNA levels in tissues of rhesus macaques after penile SIV inoculation. (A, C, E, and G) The level of unspliced vRNA, representing gRNA in virions and infected cells and the bulk of the SIV mRNA in infected cells, is shown; (B, D, F, and H) spliced vRNA is only a fraction of the SIV mRNA in infected cells, but it is an unambiguous marker of transcription from integrated SIV DNA provirus. Tissues were collected on the indicated days postinoculation. The animal number associated with each symbol is indicated. The dashed vertical lines divide the tissues into 4 anatomic regions. From left to right, the regions are (i) regions of the penis, (ii) genital lymph nodes, (iii) systemic lymphoid tissues, and (iv) gastrointestinal tract and draining mesenteric LN. Obt LN, obturator LN; Ing LN, inguinal LN; Ax LN, axillary LN; Mes, mesenteric LN.

FIG 3

SIV RNA⁺ cells in inguinal LN and foreskin. Cells containing vRNA (brown) were detected in tissue sections using in situ hybridization with antisense SIV-specific riboprobes. The interval between the time of SIV inoculation and the time of tissue collection is indicated for each row. (A, B, and E to H) Sections of tissue from animals inoculated with SIVmac251 hybridized with antisense SIV-specific riboprobes. (C and D) Sections of tissue from an animal inoculated with AT-2-inactivated SIV and necropsied at 1 day p.i. (C) A normally processed section hybridized with antisense SIV-specific riboprobes. (D) RNase treatment prior to hybridization with antisense SIV-specific riboprobes. Arrowheads in panel C, areas in the section with abundant vRNA staining in a faint reticular pattern that is consistent with extracellular SIV virion RNA; arrowheads in panel G, 3 of the few SIV RNA⁺ cells within the T cell zone of the LN. Bars = 20 μm (A to E), 50 μm (F, H), and 100 μm (G). A DAB label and hematoxylin counterstain were used.

FIG 4

SIV RNA⁺ cells in the penis and colon. The interval between the time of SIV inoculation and the time of tissue collection is indicated for each row. All panels show sections of tissues collected from animals inoculated with SIVmac251 hybridized with antisense SIV-specific riboprobes. Arrows, representative vRNA⁺ cells in each section. Bars = 20 μm (A, B), 50 μm (C), and 100 μm (D). A DAB label and hematoxylin counterstain were used.

FIG 5

Immunophenotype of SIV RNA⁺ cells 24 h after SIVmac251 inoculation. Cells containing vRNA were detected in tissue sections using in situ hybridization with fluorescently tagged antisense SIV-specific riboprobes and antibodies to phenotype cells using cell markers in tissues at 24 h p.i. (A) Inguinal lymph node; (B) spleen. Solid arrows, representative SIV RNA⁺ T cells (bright blue); these are often associated with p55⁺ (fascin-positive) DCs; dashed arrow in panel A, a vRNA⁺ T cell abutting a p55⁺ DC; circles with dashed lines in panel B, vRNA within macrophages. The pattern of vRNA in the macrophage cytoplasm (several discrete vRNA⁺ foci) is in a pattern consistent with the phagocytosis of vRNA⁺ T cells. Red, CD3⁺ T cells; green, p55⁺ endothelial cells and bone marrow-derived DCs; yellow, CD68⁺ macrophages; bright blue, SIV RNA; dark blue, DAPI staining of nuclear DNA. Bars = 20 μm.

FIG 6

Immunophenotype of SIV RNA⁺ cells in the inguinal LN. Cells containing vRNA were detected in tissue sections using in situ hybridization with fluorescently tagged antisense SIV-specific riboprobes and antibodies to phenotype cells using cell markers. The images are from 1 day p.i. (A and B), 7 days p.i. (C), and 14 days p.i. (D) (A, C, D) Tissues collected from animals inoculated with SIVmac251; (B) tissue collected from an animal inoculated with AT-2-inactivated SIV and necropsied at 1 day p.i. Arrows, representative SIV RNA⁺ cells (bright blue) located mostly in the T cell-rich paracortex (blue); arrowheads in panel B, extracellular vRNA within a B cell follicle; arrowheads in panel D, vRNA within macrophages. The vRNA signal is separated by a clear space from the surrounding macrophage cytoplasm, as if it were in a phagosome. Red, CD3⁺ T cells; green, p55⁺ endothelial cells and bone marrow-derived DCs; yellow, CD68⁺ macrophages; bright blue, SIV RNA; dark blue, DAPI staining of nuclear DNA. Bars = 20 μm.

FIG 7

Density of SIV RNA⁺ cells in tissues. Cells containing vRNA were detected in tissue sections using in situ hybridization. The number of cells in >5 tissue sections of known surface area were counted, and the results are expressed as the number of SIV RNA⁺ cells/10 cm². Each symbol corresponds to the indicated tissue. Red, genital tract; blue, genital LNs; black, systemic lymphoid tissue (spleen); purple, GI tract. Results from the animals inoculated with AT-2-inactivated SIV are within the vertical gray columns at 1 and 3 days p.i. Note that not every tissue was assessed in every animal, but all available results are shown.