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CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, July 1995, p. 400–403 1071-412X/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 2, No. 4

Presence of Neutralizing Antibodies to Heterologous Human Immunodeficiency Virus Type 1 Isolates in Sera of Infected Individuals Is Not Predictive of Rate of Disease Progression RONALD Q. WARREN,1,2 MICHAEL T. WONG,2,3 GREGORY P. MELCHER,2,3 STEPHEN P. BLATT,2,3 INES ZAPIOLA,4 MARIA BELEN BOUZAS,4 GUILLERMO MUCHINIK,4 STEPHANIE A. ANDERSON,1,2 AND RONALD C. KENNEDY1,2* Department of Virology and Immunology1 and Center for AIDS Research,2 Southwest Foundation for Biomedical Research, San Antonio, Texas 78228; Department of Infectious Diseases, Wilford Hall USAF Medical Center, Lackland Air Force Base, Texas 782362; and Grupo de Trabajo de SIDA Pediatrico, Area Diagnostico, Hospital de Nin ˜os ‘‘Ricardo Gutierrez,’’ Buenos Aires, Argentina4 Received 7 November 1994/Returned for modification 9 December 1994/Accepted 14 March 1995

These studies were undertaken to examine whether the presence of human immunodeficiency virus type 1 (HIV-1)-neutralizing antibodies in sera of infected individuals would alter the rate of disease progression. HIV-1-infected individuals (n 5 87) were initially examined for neutralizing activity in vitro against both laboratory and tissue culture-adapted clinical heterologous HIV-1 isolates. The neutralizing activities of sera were determined by a 90% or greater reduction in HIV-1 p24 levels in vitro. In a cross-sectional analysis of all infected individuals, we observed that sera from asymptomatic individuals neutralized a significantly greater number of heterologous HIV-1 isolates than sera from symptomatic patients. Patients who could be followed up longitudinally (n 5 24) were then studied to determine the impact of neutralizing antibodies on the rate of disease progression. We observed no significant difference between the numbers of HIV-1 isolates neutralized in vitro by sera from patients who remained clinically stable and by those from patients who progressed rapidly. Our data indicated that the presence or absence of neutralizing antibodies to heterologous HIV-1 isolates was not associated with the rate of disease progression. Type-specific neutralizing antibodies appear initially after infection with human immunodeficiency virus type 1 (HIV-1), with more broadly reactive antibodies developing over time (1, 3, 14, 21). The clinical relevance of neutralizing antibodies in sera of HIV-1-infected patients is presently unclear. Several studies have suggested that a correlation may exist between the presence of neutralizing antibodies and an improved clinical status of infected patients (15, 22). For example, sera from HIV-1-infected infants who were clinically stable were found to possess neutralizing antibodies more frequently than sera from infants with disease progression (15). In addition, it has been reported that the presence of group-specific neutralizing antibodies in infected mothers lowers the risk of transmission of HIV-1 (16). However, neutralizing antibodies do not appear to prevent the development of AIDS in infected individuals, since sera from many end stage patients exhibit HIV-1-neutralizing activity in vitro (3, 21). With most viral infections, the presence of neutralizing antibodies in sera signals recovery from illness and protection against reinfection. While there are reports of chimpanzees with neutralizing antibodies being protected against experimental challenge with HIV-1, this correlation has not been shown to be consistent (2, 4, 6, 7, 13). Our initial study examined 87 randomly selected HIV-1infected individuals, who were clinically grouped as symptomatic or asymptomatic for HIV-1-induced disease. A cross-sec-

tional analysis compared the HIV-1-neutralizing activities of sera from asymptomatic and symptomatic patients against four heterologous HIV-1 isolates, consisting of two laboratory strains and two clinical isolates obtained from AIDS patients. Individuals who could be followed up longitudinally (n 5 24) were then examined to determine whether the presence or absence of neutralizing antibodies in sera altered the rate of disease progression. Sera from HIV-1-infected individuals from both the United States and Argentina were examined in this study. We have previously screened these sera for antibody reactivity against a panel of V3-based peptides by enzyme-linked immunosorbent assay (ELISA). We found that sera from the two countries had similar binding patterns, suggesting that both groups of individuals were exposed to similar populations of HIV-1 subgroup B isolates (20). The data presented in this paper indicate that sera from asymptomatic individuals neutralize a greater number of HIV-1 isolates than sera from symptomatic individuals. Decreased neutralizing activity of sera from symptomatic individuals against both laboratory and clinical HIV-1 isolates was observed. These findings extend our previous observations that antibody recognition of selected HIV-1 gp160 epitopes narrows in reactivity with disease progression (19, 23). Longitudinal analysis indicated that the presence or absence of neutralizing antibodies against heterologous HIV-1 isolates did not significantly affect the rate of disease progression.

* Corresponding author. Present address: Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, OK 73190. Phone: (405) 2712133. Fax: (405) 271-3117.

MATERIALS AND METHODS HIV-1-infected cohort. Serum specimens from HIV-1-infected individuals from the United States (n 5 37) and Argentina (n 5 50) were examined in this

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TABLE 1. Mean number of HIV-1 isolates neutralizeda

TABLE 2. Number of HIV-1 isolates neutralized by sera from infected individualsa

Mean (SD) no. neutralized in vitro Patient group (n)

Asymptomatic (48) Symptomatic (39)

Total isolates

Laboratory isolates

Clinical isolates

2.4 (1.1) 1.7b (1.2)

1.5 (0.7) 1.0b (0.9)

1.0 (0.5) 0.7b (0.5)

a Neutralization was determined by a 90% or greater reduction in p24 levels. The minimum dilution of sera tested for neutralizing activity was 1:50. Sera were tested against two laboratory (LAI and SF2) and two clinical (T5G10 and A284) HIV-1 isolates. b Represents a statistically significant decline in mean number of isolates neutralized compared with the result for asymptomatic individuals (P , 0.01).

study. All individuals were confirmed to be seropositive to HIV-1 by Western blot (immunoblot) analysis (Bio-Rad Laboratories, Richmond, Calif.). Serum specimens from the United States were obtained from active-duty military personnel, their dependents, and retirees at Wilford Hall USAF Medical Center, Lackland Air Force Base, Tex. The group of subjects from the United States consisted of 36 males and 1 female and of 23 asymptomatic and 14 symptomatic individuals. The designation of individuals as symptomatic or asymptomatic was determined according to the World Health Organization staging system (24). The 24 individuals examined longitudinally for the effect of neutralizing antibodies on the rate of disease progression were from the United States. These individuals were selected because sera and clinical information, such as CD41 cell levels, spanning a period of at least 12 months were available. All individuals examined longitudinally had CD4 cell levels measured at Wilford Hall USAF Medical Center. Specimens from Argentina were obtained from HIV-1-infected individuals examined in Buenos Aires. This group consisted of 37 males and 13 females. One-half of the infected individuals from Argentina were symptomatic. Ten sera from healthy, uninfected individuals from both the United States and Argentina were used as controls. All serum samples were heat inactivated at 568C for 30 min prior to use. Neutralization assays. Sera from both the United States and Argentina were examined in a cross-sectional manner for HIV-1-neutralizing activity in vitro. A reduction in p24 levels, determined by a p24 antigen capture ELISA, was used to show neutralizing activity of sera against the HIV-1 isolates (Retrovirology Coulter Corp., Hialeah, Fla.). This procedure has been described in detail previously (18). One hundred 50% tissue culture infectious doses of each virus were added to microtiter wells containing SupT1 cells, together with test sera. Infected culture supernatants were diluted to obtain optical density values in the linear region of the p24 standard curve, and the concentrations of p24 in the culture supernatants were determined. Sera were considered to contain neutralizing activity if a reduction in p24 levels of greater than 90% compared with the levels in wells containing virus plus normal sera was detected. Neutralization titers were expressed as the arithmetic mean titer and were determined by utilizing doubling dilutions of sera. Positive titers were determined by calculating the greatest dilution of serum that inhibited p24 levels by more than 90% compared with control serum. HIV-1 isolates. Two HIV-1 clinical isolates were utilized in this study. These were obtained from an infected adult AIDS patient from Tanzania (isolate T5G10) and an infected 4-month-old child from Argentina (isolate A284). In both instances, virus was obtained by coculturing HIV-1-infected peripheral blood mononuclear cells (PBMCs) with normal phytohemagglutinin-stimulated PBMCs. Viral cultures were expanded by infecting SupT1 cells with medium from the PBMC cocultures. Medium from the SupT1 cells was aliquoted and stored in liquid nitrogen. On the basis of partial sequence analysis and serologic reactivity, both of these isolates appeared to belong to HIV-1 subgroup B. The laboratory isolate LAI (IIIB) was obtained from the AIDS Reference Repository, Division of AIDS, National Institute of Allergy and Infectious Diseases, while SF2 was obtained from Jay Levy, University of California, San Francisco. Statistical analysis. An analysis of the number of HIV-1 isolates neutralized by sera from infected individuals was performed by utilizing Student’s t test. Mean titers of neutralizing antibodies in sera from individuals grouped as stable or as rapid progressors were compared by Student’s t test for the difference between two group means.

RESULTS Cross-sectional analysis of neutralizing activity. Sera from HIV-1-infected individuals, grouped as asymptomatic or symptomatic, were tested for neutralizing activity against four HIV-1 isolates in vitro (Table 1). Two of the isolates were laboratory strains (LAI and SF2), while two were clinical isolates obtained from infected human PBMCs adapted to tissue culture and utilized to infect T-cell lines (T5G10 and A284).

401

Patient group (n)

Asymptomatic (48) Symptomatic (39) a

No. (%) of sera with neutralizing activity for the following no. of isolates neutralized: 4

3

2

1

0

6 (13) 0 (0)

21 (44) 14 (36)

11 (23) 10 (26)

7 (15) 5 (13)

3 (6) 10 (26)

The minimum dilution of sera tested for neutralizing activity was 1:50.

We observed that sera from asymptomatic individuals neutralized a significantly greater number of isolates than sera from symptomatic individuals (P , 0.01). This increased neutralizing activity of sera from asymptomatic individuals was observed with both laboratory and clinical isolates. We observed that the majority of sera from asymptomatic individuals neutralized three or more isolates (57%), compared with 36% of the sera from symptomatic individuals (Table 2). Longitudinal analysis of disease progression. Longitudinal CD4 cell values were available for 24 individuals and were monitored for a period of 12 to 36 months following the initial evaluation for neutralizing antibodies. These individuals were examined in order to determine whether the presence of neutralizing antibodies to heterologous HIV-1 isolates would have a beneficial effect on the rate of disease progression. Changes in levels of CD41 cells in the peripheral blood were used as an indicator for disease progression. Infected individuals were grouped according to their rate of decline of CD41 cells in the peripheral blood over the 12- to 36-month time span. These groups consisted of individuals whose CD4 cell levels declined by ,20% (stable), 20 to 50% (intermediate), and .50% (rapid) during the 12- to 36-month period (Table 3). When sera from the three groups were compared for the number of HIV-1 isolates neutralized in vitro, no significant difference was detected. Sera from individuals who remained clinically stable neutralized from one to four of the HIV-1 isolates (mean 5 3.0) in vitro. Similar patterns of neutralizing activity against heterologous HIV-1 isolates were also observed for sera from individuals who exhibited either intermediate or rapid rates of disease progression (mean number of isolates neutralized 5 3.0 and 2.75, respectively). We were unable to demonstrate a beneficial impact of heterologous HIV-1-neutralizing antibodies in sera of infected individuals on the rate of disease progression. Six selected individuals (Table 3) were followed up longitudinally for changes in CD4 cell levels over a time period from their initial clinical evaluation to their most recent assessment (Fig. 1). The approximate date of infection has not been determined for these individuals. While some individuals maintained relatively stable CD4 cell levels over extended periods of time (Fig. 1a), others exhibited dramatic declines in CD4 cell levels (Fig. 1b). Initial serum samples from these individuals demonstrated similar levels of neutralizing activity, neutralizing two to four of the HIV-1 isolates in vitro. Endpoint neutralization titers. We also examined antibody titers against the four HIV-1 isolates for six individuals classified either as stable or as rapid progressors. This was done to rule out the possibility that differences in the rates of disease progression might reflect differences in neutralizing-antibody titers. The endpoint neutralization titers against most HIV-1 isolates were two- to fourfold greater in sera from the clinically stable group (range, 1/50 to 1/800) than in sera from the rapid progressors (range, 1/50 to 1/400) (Table 4). However, the mean endpoint titers of neutralizing antibodies for these six

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TABLE 3. Change in CD4 cell levels following evaluation of sera from infected individuals for HIV-1-neutralizing activity Rate of disease progressiona and patient no.

Stable 1 2 3 4 5 6 7 8 9 10 11 Intermediate 12 13 14 15 16 Rapid 17 18 19 20 21 22 23 24

% Decline in CD4 cell levelb

No. of HIV-1 isolates neutralizedc

0 0 0 0 15 0 2 0 0 5 18

4 2 3 3 3 3 1 4 4 3 3

25 42 43 26 20

4 3 3 2 3

51 86 62 78 98 92 86 89

4 3 3 3 3 3 2 1

a Individuals were grouped according to their rate of decline in CD41 cells in the peripheral blood over a 12- to 36-month period. Individuals classified as stable or as having intermediate or rapid progression exhibited declines in CD4 cells of ,20%, 20 to 50%, and .50%, respectively. b Determined by comparing the last CD4 cell value with the initial value obtained during the evaluation for neutralizing activity. c The HIV-1 isolates examined were the same as for Table 1.

individuals were not found to be significantly different (P 5 0.17).

FIG. 1. Longitudinal analysis of CD41 cell levels in patients grouped as stable (a) or rapid progressors (b). All sera were tested for neutralizing activity at the 0-month time point.

DISCUSSION Serious questions currently exist concerning the effectiveness of the humoral immune response to HIV-1 in humans. While infected individuals typically mount a strong humoral response to HIV-1 envelope glycoproteins, their ability to control viral proliferation appears to be limited (19). Neutralizing antibodies directed against autologous isolates appear shortly after infection with HIV-1 and may play a role in suppressing viral proliferation. However, the rapid appearance of HIV-1 escape mutants limits the effectiveness of these antibodies (1, 10). In addition, more broadly reactive neutralizing antibodies reportedly develop several months to years following infection (5, 8, 12). Despite the presence of these antibodies, viral proliferation reportedly continues, with patients eventually developing AIDS (11, 17). The data presented in this paper suggest that sera from asymptomatic individuals neutralize a significantly higher number of heterologous HIV-1 isolates in vitro than sera from symptomatic patients. Neutralizing antibodies with cross-reactivity to multiple HIV-1 isolates are apparently lost as individuals become symptomatic. We observed less neutralizing activity by sera from symptomatic patients against both laboratory and clinical HIV-1 isolates.

When infected individuals were followed up longitudinally, we observed no significant correlation between the presence of neutralizing antibodies in sera and the rate of disease progression over a period of 12 to 36 months. The period of 12 to 36 months was selected because follow-up times for each individual varied from 1 to 8 years. By examining individuals over a defined period of time, a more meaningful comparison of the

TABLE 4. HIV-1 endpoint neutralization titers of sera from individuals grouped as clinically stable or as rapid progressors Rate of disease progression

Patient no.

Reciprocal endpoint neutralization titer against HIV-1 isolate: LAI

Stable Rapid

a

Neg, negative.

2 10 11 17 22 23

50 100 100 50 50 Neg

SF2 a

Neg 100 100 50 100 100

A284

T5G10

Neg Neg Neg 50 Neg Neg

200 800 400 100 400 100

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effect of neutralizing antibodies on the rate of disease progression could be performed. Some individuals, with neutralizing activity against three or more isolates, were found to have relatively stable CD4 cell levels during this time period. However, others with similar initial levels of neutralizing activity exhibited rapidly declining CD4 cell levels. When HIV-1 neutralization titers of individuals with stable and rapidly declining CD4 cell levels were compared, asymptomatic individuals exhibited higher neutralizing titers; however, no statistically significant differences were observed. It therefore appears that while neutralizing antibodies against heterologous HIV-1 isolates are more commonly found in sera of asymptomatic individuals, they are not useful indicators for predicting the rate of disease progression. The rate of disease progression was observed to vary independently of the number of HIV-1 isolates neutralized in vitro by these sera and of their endpoint neutralization titers. The presence of neutralizing antibodies against autologous HIV-1 isolates may potentially have more prognostic value. For example, a report by Lu and colleagues (9) indicated that the presence of high titers of neutralizing antibodies against autologous viral isolates was associated with a lack of disease progression, while low titers correlated with rapid progression. However, the potential for additional HIV-1 variants to emerge during the period of time needed to isolate the autologous virus may limit the clinical value of autologous neutralization assays (1, 10). We have previously observed, in both cross-sectional and longitudinal analyses of sera from HIV-1-infected individuals, that antibodies to specific gp160 epitopes are lost as CD4 cell levels decline (19, 23). In addition, we have reported that sera from asymptomatic individuals from the United States reacted with a greater number of V3-based peptides than sera from symptomatic individuals (18). These previous studies suggest that the humoral immune system targets fewer and fewer HIV-1 gp160 epitopes (and, consequently, viral isolates) as CD4 cell levels decline. Thus, the loss of group-specific neutralizing antibodies in sera from symptomatic individuals appears to be associated with a narrowing of the antibody response during disease progression. In summary, it appears that the reactivity of HIV-1-neutralizing antibodies in sera of infected individuals narrows as individuals become symptomatic. This loss of neutralizing activity in sera from symptomatic individuals appears to coincide with the loss of antibody reactivity to specific gp160 epitopes as CD4 cell levels decline. In addition, neither the presence in sera of neutralizing antibodies against heterologous HIV-1 isolates nor their titer was associated with the rate of disease progression. ACKNOWLEDGMENTS

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