Serial passage of HIV-2UC2 using adoptive transfer of whole blood and bone marrow aspirates.
Adoptive transfer of bone marrow cells during the acute phase of infection has been shown to enhance the virulence of SIV and SHIV isolates (24
). We therefore first inoculated 10,000 TCID50
into baboon 12281 by the intravenous route. After 3 months, 10 ml of blood and 3 ml of bone marrow aspirate were transferred into a naive baboon (animal 10568). Four additional serial passages were then performed in naive juvenile baboons over the course of ca. 1 year (Fig. ).
FIG. 1. Serial passage of HIV-2UC-2 isolates in baboons. Samples (10 ml of whole blood and 3 ml of bone marrow aspirates) were taken from baboons infected with HIV-2UC2 only. The first uninfected baboon (animal 12281) received 10,000 TCID50 of HIV-2UC2/9429 isolated (more ...)
In five of the six HIV-2-inoculated baboons, persistent virus isolation from PBMC was achieved. Moreover, all of these baboons developed lymphadenopathy during the first few weeks of infection. In addition, numerous clinical signs of disease were observed, including circulating reactive lymphocytes, thrombocytopenia, alopecia, diarrhea, and gingivitis. A summary of the clinical findings and virus isolation is presented in Table . One of the baboons that developed diarrhea (animal 11966) was also infected with whipworm (Trichuris trichiura) and Balantidium coli. Another baboon that developed diarrhea was infected with the intestinal parasites Entamoeba coli and Iodoamoeba butschlii. These are not opportunistic infections, but these parasites were only observed in association with lymphadenopathy during the acute stage of infection.
Clinical findings and virus isolation in HIV-2-infected baboons after serial passage of virus in whole blood and bone marrowa
After the fourth and fifth serial passages of virus in blood and bone marrow aspirates, the two baboons (animals 12741 and 12933) had a CD4+-cell decline (321 and 269 CD4+ cells/μl of whole blood) between weeks 2 and 8 postinoculation and did not recover their preinfection levels (CD4+ cells at 800 to 1,400 cells/μl; data not shown). These findings suggest that the animals over time might progress to disease. At 2 weeks postinoculation, both of these baboons also showed a dramatic rise in the number of CD8+ cells (from 350 CD8+ cells/μl at the time of inoculation to 2,036 and 1,255 CD8+ cells/μl for baboons 12741 and 12933, respectively). After 1 year of HIV-2 infection, the ratio of CD4+ to CD8+ T lymphocytes in the majority of the baboons was <0.5:1. Before HIV-2 inoculation, the CD4+/CD8+ T-lymphocyte ratio was ca. 1:1. No substantial changes were noted in other white blood cell subsets. The overall antibody responses to HIV-2 gp120 ranged from 1:1,000 to 1:10,000 in the baboons inoculated with HIV-2 via blood and bone marrow (data not shown).
Serial passage of HIV-2 increased viral loads in plasma, PBMC, and lymphatic tissues.
Plasma viral loads in the baboons were increased as measured by quantitative-competitive PCR. These loads ranged from 50,000 viral RNA copies per ml of plasma at 2 weeks postinoculation at the first serial passage (baboon 10568) to 200,000 viral RNA copies per ml of plasma at the fifth serial passage (animal 12933; Fig. ). The viral loads in these baboons in plasma reached a set point at between 1,000 (baboons 10568, 11999, and 11966, passages 1 through 3) and 18,000 (baboon 12741, passage 4) viral RNA copies/ml at 20 weeks postinoculation.
FIG. 2. RNA viral loads in plasma in HIV-2-infected baboons inoculated with serial passages in virus in blood and bone marrow. HIV-2 RNA was measured by quantitative PCR amplification of the long terminal repeat region. The highest levels of plasma viremia were (more ...)
To determine the number of HIV-2-infected PBMC in each of the baboons, infected cell titration was performed. As shown in Table , the number of HIV-2-infected PBMC was similar in each of the baboons during the acute phase of infection (between 10,000 and 10 HIV-2-infected cells per 106 PBMC at 2 to 12 weeks postinoculation, respectively). However, 1 year postinoculation, three of the baboons that received blood and bone marrow at the later serial passages (baboons 11966, 12741, and 12933) had a substantial increase in the PBMC-associated viral load (between 100 and 10,000 HIV-2-infected cells per 106 PBMC at 52 weeks postinoculation). In addition, these baboons had substantially higher viral loads within their lymphatic tissues than the baboons receiving earlier passages (104 HIV-2-infected cells per 106 total cells versus 1 to 102 HIV-2-infected cells per 106 total cells in the spleen, tonsils, and adenoids from baboons in the first serial passage) (Table ).
Viral load in PBMC of baboons after serial passage of virus in whole blood and bone marrow transfera
Viral load in baboon PBMC and lymphatic tissues after serial passage of virus in blood and bone marrow from HIV-2-infected baboonsa
The lymphatic tissues that had the highest viral loads were the tonsils, cervical lymph nodes, and the spleen (Table ). The oral mucosal-associated lymphatic tissue has been shown to be an active site both for chronic HIV-1 infection (15
) and primary SIV infection (41
). The present studies indicated it was also an efficient anatomic site for HIV-2 replication in baboons (see below). The higher viral load in the spleen may reflect viral particle trapping rather than active virus replication although the spleen of one baboon contained a large amount of RNA+
cells and did not show viral particle trapping by in situ hybridization (see below).
Lymphoid tissue pathogenesis.
A hallmark of lymphadenopathy in HIV infection is the breakdown of the germinal centers in lymph nodes known as follicular lysis (35
). This finding can occur in a relatively early phase of HIV infection and corresponds to dendritic trapping of HIV particles in the germinal center. Later in the disease there is generally lymphoid depletion. We have observed such lymphoid depletion in HIV-2-infected baboons with an AIDS-like illness (3
). In the baboons receiving serial passages of HIV-2, the beginning of lymphoid tissue disruption was observed with follicular lysis at 6 months postinoculation. At the time of necropsy, the most pronounced HIV-related disease was observed after three serial passages in baboons 11966 and 12933) (Table ). In these HIV-2UC2
inoculated baboons, widespread vascular and endothelial cell proliferation, follicular lysis, and lymphoid depletion were observed. The process was generalized because florid hyperplasia was readily observed in many of the lymphoid tissues from each of the baboons. Moreover, fibrosis, hyalinization, and eosinophilic infiltrates were also observed.
HIV-associated pathogenesis in lymphatic tissues of HIV-2UC2-infected baboons after serial passage of virus in blood and bone marrowa
Virus replication in lymphatic tissues.
To assess viral replication within lymphatic tissues, in situ hybridization was performed on two of the HIV-2UC2-inoculated animals, baboons 12281 and 10568 (data not shown). Active virus replication was found primarily within the germinal center, as well as in the paracortical T-cell zones in peripheral lymph nodes and in the marginal sinus lying beneath the capsule. In the spleen, active virus replication and very fine viral particle trapping was present in the germinal center. In contrast, the gut-associated lymphoid tissue, including the Peyer's patches, showed virus replication within the germinal center without viral particle trapping. In the tonsil tissues, HIV-2-infected cells were seen throughout the germinal centers and the extrafollicular regions. In addition, virus replication was noted in the lymphoepithelium of the tonsil near the lumen. No virus infection was detected in the liver, kidney, heart, pancreas, adrenal gland, lamina propia of the intestine, or ovary. However, virus replication was observed in four cells in the testis of baboon 10568.
To determine the extent of HIV-2-infection within the lymphoid tissues, we counted the number of HIV-2-infected cells, measured the area of the section and calculated the frequency of RNA+ cells per mm2 of tissue section (Table ). The highest number of infected cells was found in baboon 10568 that had received HIV-2 after serial passage. Most of the virus replication was present within the tonsils, supporting our viral load determination by infection center assay. The number of virus-infected cells was highest in the T-cell zone, followed by the germinal centers and finally in the sinuses of the lymph node. A large number of these tissues also exhibited germinal center trapping of virus particles.
HIV-2-infected cells in lymphatic tissues from baboons after serial passage of virus in whole blood and bone marrowa
Changes in the biological phenotype of HIV-2.
We found that HIV-2UC2/12741, recovered from the fourth serial passage, replicated to higher levels in baboon PBMC than the original human isolate HIV-2UC2 and the HIV-2UC2/9429 isolate that was recovered from the infected baboon with an AIDS-like disease. In two separate studies, HIV-2UC2/12741 replicated more rapidly and showed peak RT activity of up to 500,000 cpm/ml of culture fluid between 7 and 10 days after inoculation in vitro. In contrast, cells infected with HIV-2UC2 had a mean RT activity of 80,000 cpm/ml of culture fluid, and those infected with HIV-2UC2/9429 had a mean RT activity of 200,000 cpm/ml. These data indicated that the virus isolated from baboon 12741, after serial passage, was better adapted to replication in baboon PBMC than the original infecting HIV-2 isolate. In addition, when inoculated onto the MT-2 T-cell line, the serially passaged isolate, HIV-2UC2/12741 was more cytotoxic than the HIV-2UC2/9429 isolate. However, human macrophages infected with HIV-2UC/12741 did not demonstrate any difference from the parental virus isolate (HIV-2UC2) in virus replication (~200,000 cpm/ml of RT activity).