CD4⁺-T-Cell and CD20⁺-B-Cell Changes Predict Rapid Disease Progression after Simian-Human Immunodeficiency Virus Infection in Macaques^{<a href="#FN151" rid="FN151" class=" fn">†</a>}

Intrarectal inoculation of 2,500 tissue culture infectious doses (TCID) is required for productive SHIV_89.6PD infection.

Male (n = 5) and female (n = 5) juvenile rhesus macaques (Maccaca mulatta) bred in captivity were housed at the Wisconsin Regional Primate Research Center (WRPRC) and used in these studies. They were confirmed negative for antibodies to SIV and type D simian retroviruses prior to this study. Macaques were immobilized with ketamine hydrochloride (10 mg/kg of body weight) before all virus inoculation and blood collections. The WRPRC is accredited by the American Academy for Laboratory Animal Care. The Institutional Animal Care and Use Committee approved all animal research protocols.

Ten rhesus macaques were inoculated intrarectally (14) with doses of SHIV_89.6PD ranging from 25,000 to 2.5 TCID. The pathogenic SHIV used in these studies was SHIV_89.6PD. This virus was derived by passaging the nonpathogenic SHIV_89.6 (17) multiple times in macaques and was isolated in CEMx174 cells with plasma from the fourth animal (K542) in this series of passages (10).

Six animals inoculated with 250 TCID or less were negative for all measures of infection for 30 weeks. These animals were then inoculated a second time with 2,500 TCID of virus by the intrarectal route, whereupon all six were persistently infected. Previous intrarectal exposure to low-dose SHIV_89.6PD did not cause transient viremia and did not affect subsequent intrarectal virus challenge. We combined data for all 10 infected animals to evaluate markers of disease progression.

Viral burden following intrarectal SHIV_89.6PD inoculation.

Infectious virus was detected in peripheral blood mononuclear cells (PBMC) from all 10 animals by 4 weeks after intrarectal inoculation with the minimal infectious dose (2,500 TCID) or a larger dose. Plasma samples from 8 of 10 animals were positive for SIV p27 antigen by 2 weeks after infection (Table ​(Table1).1). Plasma antigenemia was assessed at 1, 2, 3, 4, 6, and 8 weeks postinoculation (p.i.). We noted only a single positive sampling interval (2 weeks p.i.). The two animals with negative tests may have had a brief spike of p27 in plasma that fell between sample collections. Levels of p27 in plasma varied from <50 to 22,599 pg/ml, with an average of 5,545 pg/ml. Two macaques (r94080 and r92014) had p27 levels just below detection limits, while three macaques (r92069, r94054, and r92058) had levels of p27 greater than 11,000 pg/ml. p27 was not detected later than 2 weeks p.i., and testing was discontinued by 8 weeks p.i.

Rapid loss of greater than 90% of circulating CD4 ^{T cells in half of infected macaques.}

PBMC were purified, and lymphocyte subsets were detected as described previously (15). We collected three samples before infection to measure baseline values. All samples were within the normal range for juvenile rhesus macaques, which is 1,300 ± 420 CD4 ^{T cells/μl of blood (}19a). Nine macaques had decreases in the absolute number of circulating CD4 ^{T cells following intrarectal SHIV inoculation. CD4-T-cell loss had two general patterns: (i) rapid and dramatic loss (>90% decrease) and (ii) variable and moderate loss (35 to 80% decrease). Both outcomes were equally represented in our cohort. Five macaques had rapid and dramatic losses of circulating CD4 cells. Less than 10% of their CD4 T cells remained by 2 weeks p.i., with the average number of CD4 cells falling to 65 cells/μl of blood (Fig.} ​(Fig.1A).1A). Consistently, these five macaques had less than 10% of their CD4 ^{T cells remaining over the course of infection, with the absolute number of CD4 T cells decreasing to undetectable levels. Surprisingly, several animals survived more than 20 weeks with CD4-T-cell counts below 100 cells/μl of blood.}

FIG. 1

Changes in the number of circulating CD4 ^{T cells. The percentages of CD4 T cells remaining for weeks 1 through 24 p.i. were calculated for each animal with the following formulas: percentage of CD4 T cells remaining (at week} x) = 100 + percent change; percent change = 100 × [(number of CD4 ^{T cells at week} x − number of CD4 ^{T cells at week 0)/(number of CD4 T cells at week 0)]. Results for five animals with rapid and sustained losses of CD4 T cells (A) and for five animals with moderate but varying losses of CD4 T cell (B) are shown.}

Five other macaques had variable CD4^{-T-cell changes (Fig.} ​(Fig.1B).1B). One animal (r94056) had increased CD4^{-T-cell numbers at all times following SHIV infection. Macaque r94056 had low baseline CD4-T-cell counts (average of 609 CD4 T cells/μl of blood for two preinfection samples), but these values were well inside the normal range for juvenile rhesus macaques. By 84 days after SHIV inoculation, the absolute CD4 count for r94056 was increased to 1,882 cells/μl of blood even though the percentage of CD4 T cells in PBMC (37.1%) was unchanged from the preinfection value of 35.7%. The increase in the absolute count of CD4 T cells was due mainly to a generalized lymphocytosis, with total, absolute lymphocyte counts rising from the preinfection value of 1,707 lymphocytes/μl of blood to 5,069 lymphocytes/μl of blood by 84 days. The other unique pattern of CD4-T-cell changes was in animal r92085. An initial drop to <5% CD4 T cells by 4 weeks p.i. was followed by rising CD4-T-cell counts that reached 25% of initial CD4-T-cell counts and remained stable throughout 60 weeks of observation. Three macaques (r92014, r94080, and r94085) had average CD4 T-cell losses of 57% by 12 weeks p.i. but maintained CD4-T-cell counts above 450 cells/μl of blood. One of these three macaques experienced CD4 T-cell increases after the initial CD4-T-cell loss and ended up with CD4-T-cell numbers above the starting levels by 24 weeks p.i.}

Changes in CD20 ^{B cells.}

Changes in circulating B cells (CD20 ^{cells) were also related to disease progression (Fig.} ​(Fig.2).2). Figure ​Figure2A2A depicts CD20^{-B-cell changes in five macaques that had rapid and dramatic CD4-T-cell losses. All five of these animals had sustained losses of greater than 50% in circulating B cells. The average percentage of B-cell loss was 63% by 4 weeks p.i., with some animals losing up to 87% of their B cells.}

FIG. 2

Changes in the number of circulating CD20 ^{B cells. The percentages of CD20 B cells remaining for weeks 1 through 24 p.i. were calculated for each animal by using week 0 values as 100%. (A) Percentages of B cells remaining in the five animals with rapid and sustained losses of CD4 T cells; (B) percentages of B cells remaining in the five animals with moderate and variable losses of CD4 T cells.}

Five macaques that did not show dramatic CD4^{-T-cell losses had sustained B-cell increases after an initial drop by 1 week p.i. (Fig.} ​(Fig.2B).2B). The average B-cell increase was 105% of preinfection levels by 4 weeks after infection. Before SHIV infection, the average number of circulating B cells ± the standard deviation was 1,125 ± 888 cells/μl of blood. Eight weeks after SHIV inoculation, the average B-cell count had decreased to 390 ± 79 cells/μl of blood in the five animals with large CD4^{-T-cell losses and had increased to 1,601 ± 1,028 cells/μl of blood in the five animals with moderate CD4-T-cell losses.}

Virus-binding antibodies.

Six macaques developed virus-binding antibodies by 4 weeks after infection (Fig. ​(Fig.3).3). Plasma samples were screened on plates coated with HIV-2 antigens (Sanofi/Pasteur, Chaska, Minn.). Endpoint titers are reciprocals of the last dilution with an absorbance value greater than 0.02 after background subtraction. Three macaques had steady increases in levels of virus-binding immunoglobulin G (IgG) antibody, with peak responses occurring 16 to 20 weeks after infection. Macaque r92085 had a peak response at 16 weeks p.i., with an endpoint titer of 1:25,600. r94056 and r94085 had peak responses at 20 weeks p.i., with endpoint titers at that time of 1:6,400 and 1:25,600, respectively. A single macaque (r92081) had an initial increase of antibodies in plasma that peaked 12 weeks p.i., with an endpoint titer of 1:25,600. Antibodies in r92081 steadily declined to low, but still detectable, levels. Macaques r92014 and r94080 had moderate or low virus-binding IgG antibody responses, but these levels were stable. The endpoint titers for these animals were 1:6,400 and 1:400, respectively, at 20 weeks p.i. The remaining four animals did not produce detectable levels of virus-binding antibodies.

FIG. 3

Antibody responses in plasma. Macaque sera were tested with an HIV-2 enzyme-linked immunosorbent assay kit for antiviral antibodies. Absorbance values (405 nm) of plasma samples from weeks 0 through 24 p.i. are depicted for each animal. Open symbols represent results with seronegative macaques, and closed symbols represent results with seropositive macaques.

Plasma samples were also tested for HIV-1 env-specific IgG responses in plasma by using enzyme-linked immunosorbent assay plates coated with purified glycosylated gp120 from HIV-1_MN (provided by Ribi Immunochem, Hamilton, Mont.). Plasma samples that were positive for virus-binding antibodies (r92014, r92081, r92085, r94056, r94080, and r94085) also reacted with purified HIV-1_MN gp120, while plasma samples from all four SIV-seronegative macaques (r92058, r92069, r94054, and r94078) did not react with HIV-1_MN gp120. We did not determine strain-specific endpoint titers for envelope responses, because purified HIV-1_89.6 envelope glycoprotein was not available.

Antibodies that neutralize SHIV_89.6P in plasma.

Plasma from peak antiviral antibody responses (those from week 20 were used for all animals except r92081, for which the week 12 sample was used) were tested for the ability to neutralize SHIV_89.6P (16) in an MT-2 cell-killing assay as described previously (13, 17). The neutralizing titer reported for each animal in Table ​Table11 is the reciprocal of the plasma dilution required to protect 50% of cells from virus-induced killing. All four macaques that were negative for antibodies against HIV-1 env and SIV structural proteins in plasma were also negative for neutralizing antibodies. Macaques r94056 and r92081 had very high levels of neutralizing antibodies, with titers of 12,464 and 35,372, respectively. The four remaining seropositive macaques had moderate titers of neutralizing antibody ranging from 260 to 4,339.

Clinical outcome.

Four macaques succumbed to disease at 25, 28, 32, and 36 weeks after SHIV_89.6PD inoculation. All animals were euthanized on the advice of the veterinarian. Euthanasia was recommended when animals refused food or water, were unreactive to stimuli, or showed some severe distress. Lymphoid tissue pathology and major diagnoses at the time of necropsy provided evidence of advanced disease and supported the euthanasia decisions. Six animals remain alive (two animals at 88 weeks p.i. and four animals at 58 weeks p.i.).

r94054 (female) was euthanized 25 weeks after inoculation with 2,500 TCID. The absolute number of circulating CD4 ^{T cells decreased to below 100 CD4 cells/μl of blood within 2 weeks of virus inoculation. This animal had a sustained loss (approximately 70%) of circulating B cells and did not seroconvert before succumbing to disease. At necropsy this animal had lymphoid tissues severely depleted of lymphocytes (in follicles and in paracortex and periarteriolar lymphoid sheaths), edema (severe), cholangiohepatitis, and interstitial pneumonia. Also noted were lymphadenitis and dermatitis associated with cytomegalovirus.}

r92058 (male) was euthanized 28 weeks after inoculation with 2,500 TCID. CD4^{-T-cell counts dropped to below 10 cells/μl of blood by four weeks p.i. This animal had a sustained decrease (approximately 80% loss) in circulating B cells and remained seronegative. Major diagnoses at necropsy included mild colitis, hepatitis, and lymphocyte depletion in lymphoid tissues.}

r92069 (female) was inoculated with 25,000 TCID and was euthanized 32 weeks p.i. CD4^{-T-cell numbers decreased to 2 cells/μl of blood four weeks p.i. and remained below 10 cells/μl of blood until the time of necropsy. This animal had a decrease in B-cell numbers (<50% remaining) until 24 weeks p.i., when B-cell numbers increased to above preinfection values. r92069 did not seroconvert. The major diagnoses noted at necropsy were gastritis, interstitial pneumonia, and severe involution of lymphoid tissues.}

r94078 (female) was inoculated with 2,500 TCID and was euthanized 36 weeks p.i. Circulating CD4 ^{T cells decreased by greater than 50% by 1 week p.i. r94078 had less than 5% CD4 T cells remaining by 2 weeks p.i., with absolute numbers of CD4 T cells below 10 cells/μl of blood from 12 weeks p.i. until the time of necropsy. Follicular involution with normal or expanded paracortex and periarteriolar sheaths was noted for peripheral lymph nodes, thymus, and spleen. Severe depletion of follicles and paracortex in ileocecal and colonic lymph nodes was noted along with pyogranulomatous lymphadenitis. Interstitial pneumonia with multinucleated giant cells was present.}

Statistical analysis.

We performed two types of statistical tests to evaluate data on disease progression. The first test assessed differences in survival time as a function of laboratory markers of disease. Individual groups were identified on the basis of CD4 cell count, CD8 cell count, CD20 cell count, level of virus binding antibody, level of neutralizing antibody, and plasma antigenemia. Each marker was used to define two groups within the cohort. Animals with substantial drops in CD4 (Fig. ​(Fig.1A)1A) were grouped separately from animals with moderate drops in CD4 (Fig. ​(Fig.1B).1B). Similar groupings were developed for the other markers of infection. In each case, we divided the cohort into two groups and tested whether there was a statistically significant survival difference. These tests (Table ​(Table2)2) were performed by Kaplan-Meier analysis. Rapid and sustained CD4 loss, CD20 cell loss, a low level of virus-binding antibody, a low level of neutralizing antibody, and a high degree of plasma antigenemia (at 2 weeks) were all predictive of rapid disease progression with shortened survival. Changes in CD8 cells were not correlated with survival. This approach allowed us to identify outcomes that predicted rapid disease progression even at a time when most animals in the study remained alive.

A second statistical analysis was performed to evaluate correlations among individual markers of disease progression. Quantitative values were compared directly with Pearson’s log rank correlation coefficients, and the values for the comparisons are listed in Table ​Table3).3). Changes in CD4 cells were positively correlated with either changes in CD8 cells or changes in CD20 cells or levels of virus-binding antibody. Changes in CD8 or CD20 cells were positively correlated with levels of virus-binding antibodies. No other correlations reached statistical significance (P ≤ 0.05). It is interesting that changes in CD4 cell counts were more strongly correlated (0.789, P ≤ 0.01) with levels of virus-binding antibody than were changes in CD20 cell counts (0.659, P ≤ 0.05). Even though this difference is small, the data imply that early changes in CD4 cell populations may be important in the host’s subsequent inability to produce measurable levels of virus-binding antibody. The proportion of CD20 cells remaining by 8 to 24 weeks after inoculation was strongly correlated (P ≤ 0.01) with the level of CD4 cell loss during the acute infection interval from 2 to 4 weeks after inoculation.

This work was supported by WRPRC grant RR00167 and PHS grants AI36643 and AI38491 (C.D.P.).

We gratefully thank Kathy Schell and Kristen Elmer for assistance with flow cytometry.