Correlation of acute humoral response with brain virus burden and survival time in pig-tailed macaques infected with the neurovirulent simian immunodeficiency virus SIVsmmFGb

Abstract

Infection of pig-tailed macaques with the simian immunodeficiency virus (SIV) isolate SIVsmmFGb frequently results in SIV encephalitis (SIVE) in addition to immunodeficiency and acquired immune deficiency syndrome. We used in situ hybridization to quantitate the number of SIV-infected cells in brain parenchyma, choroid plexus, and meninges from 17 macaques that developed acquired immune deficiency syndrome after infection with SIVsmmFGb. SIV-infected cells and histopathological lesions of SIVE were identified in 15 of 17 animals (88.2%), including 12 of 12 rapid progressors (RP) and 3 of 5 slow progressors (SP). The parenchymal virus burden was much greater in RP macaques than in the three SP macaques with SIVE (median values of 24.3 versus 0.3 infected cells/mm(2), respectively; P < 0.05). Viral load differences between RP and SP with SIVE were less marked in choroid plexus (29.6 versus 12.8 infected cells/mm(2), respectively) and meninges (133.0 versus 34.2 infected cells/mm(2), respectively). A significant negative correlation was observed between the magnitude of the anti-SIV antibody titer at 1 month after inoculation and brain virus burden at necropsy (r = -0.614; P < 0.01). The close association between immune response and SIVE in this model should prove useful for identifying correlates of immune protection against primate lentiviral encephalitis.

Figure 1

Group median values for percentages and absolute numbers of CD3⁺/CD4⁺ and CD3⁺/CD8⁺ T lymphocytes in RP (n = 12) versus SP (n = 5) pig-tailed macaques before inoculation (baseline) with SIVsmmFGb and at necropsy after the development of SAIDS. In each panel, columns depict the group median value and error bars show the semi-interquartile range for the group. Percentages (A) and absolute numbers, in T cells per μl (B), of CD3⁺/CD4⁺ T lymphocytes in RP and SP macaques. Percentages (C) and absolute numbers, in T cells per μl (D), of CD3⁺/CD8⁺ T lymphocytes in RP and SP macaques.

Figure 2

Plasma viral load (A) and anti-SIV-binding antibody response (B) at necropsy of RP (n = 12) and SP (n = 5) pig-tailed macaques infected with SIVsmmFGb. A: Scatter plot showing the range of values for plasma viral load at necropsy, reported as log₁₀ SIV RNA copies/ml, for RP (•) and SP (○) groups. B: Scatter plot showing the range of anti-viral antibody titers at necropsy, reported as the reciprocal log of the last positive serum dilution, for RP (♦) and SP (⋄) groups. Horizontal bars indicate the group median value in each plot (A, B).

Figure 3

Neuropathological lesions in pig-tailed macaques infected with SIVsmmFGb. A: In situ hybridization for SIV RNA combined with H&E counterstain (in situ hybridization-SIV/H&E), demonstrating several SIV-infected cells (dark blue) within a perivascular cuff in the midfrontal cortex of macaque PMi1. Arrows identify two SIV-positive multinucleated giant cells. B: H&E-stained section showing a multinucleated giant cell in the globus pallidus of macaque PDf1. Inset a: Multinucleated giant cells are macrophage phenotype, confirmed here by IHC for CD68 (IHC-CD68, brown), and are universally infected with SIV, as shown by in situ hybridization for viral RNA in inset b (ISH-SIV, dark blue). C: H&E-stained section of midbrain from macaque PEj1, showing a microglial nodule (MGN, arrow) within the trochlear nucleus. Inset a: MGNs are focal, noncircumscribed lesions composed of heterogeneous populations of macrophage-lineage cells, as demonstrated on this serial section by IHC for CD68 (IHC-CD68, brown). Inset b: Many of the cells within MGNs are infected with SIV, as shown by in situ hybridization for SIV RNA on this serial section (ISH-SIV, dark blue). D: H&E-stained section showing a granuloma within the midfrontal cortical gray matter of macaque PSc1. In contrast to MGNs, granulomas are well circumscribed and are composed of relatively homogeneous populations of large, CD68-positive, epitheloid macrophages with abundant foamy cytoplasm, as shown in inset a (IHC-CD68, brown). Most of the macrophages within granulomas are SIV-positive, as shown by in situ hybridization for SIV RNA in inset b (ISH-SIV, dark blue). E: Choriomeningitis was observed in all SIVsmmFGb-infected macaques with SIVE. This H&E-stained section of prefrontal cortex from SP macaque POf1 shows a cellular infiltrate in the leptomeninges that includes numerous multinucleated giant cells. F: In situ hybridization for SIV RNA in a section of choroid plexus from macaque PEj1. SIV-infected cells (dark blue) can be seen within the stroma of the choroid plexus and tela choroidea. Scale bars: 50 μm (A, B, D, E, and insets in B and D); 100 μm (C, F, and insets in C).

Figure 4

In situ hybridization for SIV RNA, showing the distribution pattern of productively infected cells (dark blue) in the frontal cortex of two SIVsmmFGb-infected RP pig-tailed macaques. SIV-positive cells occur primarily in perivascular locations in SP macaques and RP macaques with low brain virus burdens (A: macaque PBf1). In contrast, infected cells were distributed diffusely throughout the parenchyma as well as around vessels in the brains of RP macaques with high brain virus burdens (B: macaque PDf1). Arrows identify blood vessels. Scale bars, 100 μm.

Figure 5

Longitudinal plots of plasma viral load (A) and anti-SIV-binding antibody response (B) in RP (n = 12), SP macaques with SIVE (SP + SIVE; n = 3), and SP macaques without SIVE (SP − SIVE; n = 2). Plasma viral load and anti-SIV antibody titer were measured at 1 and 2 months after inoculation and at necropsy. A: Plasma viral load, reported as log₁₀ SIV RNA copies/ml, for RPs (black line), SPs with SIVE (SP + SIVE; red line), and SP without SIVE (SP − SIVE; red dashed line). B: Anti-SIV-binding antibody titers, reported as the reciprocal log of the last positive serum dilution for RP (black line), SP + SIVE (red line), and SP − SIVE (red dashed line) macaques. The horizontal dashed line represents the lower limit of detection for the assay (1:400 dilution of plasma; 2.6 log₁₀ titer⁻¹).

Figure 6

Number of CD68⁺ cells in the white matter tracts of the midfrontal cortex is positively correlated with brain viral load and severity of encephalitis. A: The mean number of macrophages in midfrontal cortical white matter was determined for each SIVsmmFGb-infected macaque and three SIV-negative controls by IHC for CD68 followed by computer image analysis of 10 contiguous sections of tissue at ×200. Columns depict the mean number of CD68⁺ cells per mm² for individual macaques and error bars show the SD. Black columns represent macaques with SIVE, as defined by the presence of viral nucleic acids by in situ hybridization; gray columns represent macaques without SIVE. B: The number of CD68⁺ cells in the white matter tracts of the midfrontal cortex of RP and SP SIVsmmFGb-infected macaques is positively correlated with both brain virus burden, measured as SIV⁺ cells per mm² (P < 0.01), and the severity of SIVE, as determined by SIVE index (P < 0.01). C: The positive correlations observed between number of CD68⁺ cells in the cortical white matter and both brain virus burden and severity of SIVE are maintained when the analysis is restricted to RP macaques alone (P < 0.01).

Figure 7

Macrophages were identified by IHC for CD68 (IHC-CD68) in the white matter tracts of the midfrontal cerebral cortex in SIVsmmFGb-infected and uninfected pig-tailed macaques. Large numbers of CD68-positive cells (dark brown) were found throughout the brain parenchyma as well as in perivascular locations in RP macaques (A: RP macaque PDf1). CD68-reactive cells were cytomorphologically diverse, and included club-shaped cells, multinucleated giant cells, and cells with extensive cytoplasmic processes. In contrast, cells expressing CD68 were far fewer in number and were predominantly confined to perivascular locations in SP macaques with SIVE (B: SP/SIVE macaque POf1) and SP macaques without SIVE (C: SP macaque PKg1). CD68-positive cells were infrequently observed in SIV-negative controls (D: SIV-negative macaque PSs). Arrows identify blood vessels (B, C). Scale bars, 50 μm.

Figure 8

The phenotype of productively infected cells was determined in brain tissue from SIVsmmFGb-infected pig-tailed macaques by dual-label histological assays that combined in situ hybridization for SIV RNA (dark blue) with IHC using antibodies that recognize antigens specific for immune cells or resident cells of the CNS parenchyma (dark brown to black). A: SIV/Ham-56: Arrows identify three SIV-infected macrophages (co-localized blue and black chromogens) in the white matter tracts of the midfrontal cortex of RP macaque PZg1, using the antibody Ham-56. Uninfected macrophages (arrowhead) are also present. B: SIV/Ham-56: Arrows identify three cells out of a group of several SIV-infected macrophages (co-localized blue and black chromogens) in the cerebral gray matter of macaque PZg1. Arrowheads identify uninfected macrophages. C: SIV/CD3: Even small SIV-positive cells (arrows, blue chromogen) are generally larger than CD3-expressing cells (arrowheads, brown chromogen) and do not co-localize CD3 antigen. D: SIV/VWF: Arrows identify two SIV-infected cells (dark blue) within the perivascular space of a blood vessel in the cerebral cortex of RP macaque PUf1. The infected cells are immediately adjacent to capillary endothelial cells (arrowheads), but do not express the endothelial cell antigen, von Willebrand factor (black). E: SIV/GFAP: Arrows identify three SIV-positive cells (dark blue) that are immediately adjacent to GFAP-positive astrocytes (arrowheads, brown-black chromogen) in the medulla of macaque PUf1. Infected cells often appear cradled between astrocyte processes, but GFAP-expressing cells (astrocytes) do not co-express viral RNA. F: SIV/NF: Arrows identify two SIV-infected cells (dark blue) adjacent to the apical dendrites of pyramidal neurons (arrowheads) in the cerebral cortex of RP macaque PZg1. Neurons are labeled with an antibody that recognizes a 200-kd neurofilament polypeptide (brown chromogen). Neurons do not co-localize hybridization signal, showing that they are not productively infected with SIV. Scale bars, 50 μm.