Abstract: The immunodeficiency-defining AIDS results from a progressive decline of CD4 cell count. We previously showed that 3S, a unique motif of the HIV-1 gp41 envelop protein, is highly conserved in HIV-1 strains and induces expression of NKp44L, rendering CD4 cells sensitive to NK killing. Here we found from a well-characterized cohort of 244 untreated HIV-1 seroconverters that high levels of anti-3S antibodies significantly delay spontaneous disease progression in the first years after seroconversion; this effect was not mediated through baseline viral load or CD4. These results could have important implications both for clinical care and better understanding of pathogenicity.
*INSERM Laboratory of Immunity and Infection, UMR-S 945, University Pierre et Marie Curie, Paris, France
†AP-HP, Pitié-Salpêtrière Hospital, Paris, France
‡InnaVirVax, Evry, France
§INSERM, Center for Research in Epidemiology and Population Health, U1018, HIV Epidemiology, University Paris-Sud, AP-HP, Bicêtre Hospital, Epidemiology and Public Health Service, Le Kremlin Bicêtre, France.
Correspondence to: Vincent Vieillard, PhD, INSERM UMR-S 945, Laboratory of Immunity and Infection, Pitié-Salpêtrière Hospital, F-75651 Paris, France (e-mail: firstname.lastname@example.org).
Supported by grants from the Agence Nationale de la Recherche (ANR) (THERVAX, Grant number: P007837) and by the Agence Nationale de Recherches sur le Sida et les hépatites virales (ANRS).
The author V. Vieillard is researcher from the Centre National de la Recherche Scientifique, Crouzet J is chief executive officer of InnaVirVax, and RH Fang is an employee of InnaVirVax.
The authors have no other conflicts of interest to disclose.
Received March 14, 2012
Accepted June 1, 2012
The substantial variation of the rate of HIV disease progression among individuals complicates the uncertainty about when to initiate antiretroviral treatment.1 This variability has been largely associated to differences in set-point plasma HIV RNA levels and CD4 count; however, several publications underscore the uncertainly as to when treatment should be initiated.2–4 New correlates of disease progression in early infection, besides the routinely used viral load and CD4 count, could be invaluable for identifying the individuals most likely to benefit from early therapy.
We previously identified a highly conserved motif, called 3S, of the gp41 subunit of the HIV-1 envelop protein. During infection, this 3S motif induces expression of NKp44L, the ligand of an activating natural killer (NK) receptor, which renders CD4+ T cells sensitive to NK killing. This induction of NKp44L is highly correlated with CD4 cell depletion.5 Experiments in nonhuman primate model have confirmed that expression of NKp44L plays a key role in the pathogenesis of the disease by its association with major immunologic disturbances (altered homeostasis, increased activation, proliferation, and apoptosis of CD4+ T cells).6 Anti-3S antibodies (Ab) do not neutralize HIV-1 but were found to protect against CD4 depletion in vivo among long-term nonprogressors from the Agence Nationale de Recherches sur le Sida et les hépatites virales (ANRS) asymptomatic long-term cohort: CD4 cell depletion over a 3-year period was significantly lower in patients with detectable anti-3S Ab compared with undetectable anti-3S Ab.7 Importantly, compared with 5 other anti-gp41 Abs, only the anti-3S Ab specifically protected patients from this CD4 cell depletion.7 On the basis of these observations, we hypothesized that quantifying these specific Ab early after infection might provide additional prognostic value of subsequent disease progression. We thus studied the well-characterized ANRS SEROCO cohort of 244 untreated HIV-1 seroconvertors sampled during early HIV infection8,9; we show for the first time that high anti-3S Ab levels measured close to HIV seroconversion are associated with delayed disease progression over the following first years, independently of baseline viral load and CD4 count.
The study included 244 untreated HIV-1 seroconverters followed in the multicenter ANRS SEROCO cohort between 1988 and 1996, that is, before the combination antiretroviral therapy era.8,9 The ethics committees approved the cohort in 1987, and all subjects provided written informed consent. Eligible seroconverters had a known date of HIV-1 infection within a 24-month interval, were AIDS-free at enrollment, and had no prior antiretroviral therapy, as previously described.8,9 They were enrolled in the cohort within a median of 9 months after infection; the median follow-up in the cohort up to 1996 was 6.5 years. Frozen cells and sera were stored at enrollment and during follow-up.
ENZYME-LINKED IMMUNOSORBENT ASSAY
Anti-3S Abs were tested from the first frozen sample, collected at enrollment in the cohort. Enzyme-linked immunosorbent assay testing determined the magnitude of anti-3S Ab, as described.7 Median Ab level was 53 U/mL; 72% of the seroconverters had detectable anti-3S Ab at enrollment.
To quantify the effect of anti-3S Ab levels on the risk of spontaneous HIV disease progression, time from infection to CD4 cell count below 200 cells per cubic millimeter (91 events), clinical AIDS (83 events), and death (59 events) were estimated by using Kaplan–Meier curves that compared disease progression according to levels of anti-3S. In the final analysis, we compared subjects with Ab ≥50 U/mL (value close to the median) to the others. Kaplan–Meier curves were stratified on viral load (<4 log copies/mL vs. ≥4 log) to illustrate the specific contributions of viral load and anti-3S Abs in the risk of disease progression. Crude and adjusted relative risks (RRs) along with their 95% confidence interval (CI) were estimated by using a Cox regression model. The transient effect of Anti-3S Ab ≥50 U/mL was tested by introducing an interaction term between anti-3S Ab and time in a Cox model.
The study population of 244 seroconverters comprised mainly subjects infected through sexual route (92.7%). Twenty-two percent of the subjects were female, 66.6% were homo/bisexual men. Patients with anti-3S Ab ≥50 U/mL had lower serum viral RNA (P = 0.0004) and cellular viral DNA (P < 0.0001) levels, while CD4 cell counts did not differ between the 2 groups (Table 1).
A high level of anti-3S Ab after infection was found to be associated with delayed disease progression. From the Kaplan–Meier curves, it appeared that the protective effect was observed mainly during the first 3 years after infection (interaction term between anti-3S Ab and time: P = 0.003). Progression to CD4 levels below 200 cells per cubic millimeter (P < 0.0001) (Fig. 1A), clinical AIDS (P = 0.0005) (Fig. 1B), and death (P = 0.0008) (Fig. 1C) was delayed in the first 3 years after infection in patients with anti-3S ≥50 U/mL compared with those with values above this threshold, even when we took baseline viral load into account. The RR of a CD4 count dropping below 200 CD4 cells per cubic millimeter, estimated during the 3-year period after infection was 0.21 (95% CI: 0.07 to 0.62; P = 0.005) in patients with anti-3S ≥50 U/mL compared with the remainder. This delayed progression remained statistically significant after adjustment for age, sex, baseline CD4 count and HIV RNA (adjusted RR = 0.23, 95% CI: 0.07 to 0.73; P = 0.01). In this model, CD4 count and HIV RNA levels were also predictive of disease progression, independently of anti-3S Ab. Similar results were observed for the other endpoints, clinical AIDS and death. Sensitivity analyses using alternate cutoffs of anti-3S Ab (>60 vs. <50; or: undetectable <10, 11–85, >85) led to similar conclusions. The protective effect of high levels of anti-3S Ab was transient, likely due to the loss in antibodies. Indeed, Figure 1 clearly shows that patients with high viral loads progress faster after 3 years, irrespective of their anti-3S Ab levels. In other terms, viral load remained a strong predictor on the long term, while the effect of the anti-3S Ab was most obvious on the short-term.
A linear mixed model revealed that over the first 3 years after infection the CD4 count slope was smaller in patients with anti-3S Ab ≥50 U/mL compared with <50 U/mL, leading to mean CD4 values at 36 months after infection of 461 versus 371 CD4 cells per cubic millimeter (P < 0.004), respectively, while CD4 counts did not differ at baseline (estimated mean in the model of 611 vs. 596 CD4/mm3, respectively).
A subset of subjects had their antibodies measured again at 24–36 months after enrollment. Subjects with persistently high antibodies still had a disease-free survival advantage compared with subjects who experienced a loss in anti-3S Ab levels below 50 U/mL, but statistical significance was not reached, likely due to a smaller sample size.
To date, several serologic (β2-microglobulin, soluble CD4) markers or cell surface T cells activation (HLA-DR, CD38) have been identified as linked to the risk of progression; however, most of these predictors were not independent of viral load, a major predictor of disease progression.10,11
Our findings reveal that high levels of nonneutralizing anti-3S Ab are an independent predictor of the rate of CD4 loss, clinical HIV disease progression (AIDS) and death in the first 3 years after infection. This finding contributes to previous observations showing that not all of the interindividual variability in the rate of CD4 cell declines can be explained by viral load and confirms that other factors likely drive CD4 cell losses in HIV infection.7,12 This is observed in Sooty mangabeys and African green monkeys infected with simian immunodeficiency virus, a naturally occurring virus closely related to HIV, which show near-normal T-cell turnover and a lack of disease progression despite sustained high-level viremia.13
As shown in previous studies,6,7 the role of anti-3S Ab on disease progression is highly unlikely to just reflect higher antibody responses in general, as is observed in HIV-infected patients with better immunocompetence. The effect of anti-3S Ab in delaying spontaneous disease progression was transient, likely due to the loss in antibodies over time. In conclusion, these findings about the role of anti-3S Ab in transiently delaying HIV disease progression could have implications both in therapeutic decisions in early infection and for understanding the pathogenesis of progressive immune deterioration.
The authors thank Christine Rouzioux (Necker Hospital, Paris, France) and Jean François Delfraissy, Director of ANRS, for their contribution to the constitution of the ANRS-SEROCO cohort and the recording of clinical data.
1. Thompson MA, Aberg JA, Cahn P, et al.; International AIDS Society-USA. Antiretroviral treatment of adult HIV infection: 2010 recommendations of the International AIDS Society-USA panel. JAMA. 2010;304:321–333.
2. Sterne JA, May M, Costagliola D, et al.. Timing of initiation of antiretroviral therapy in AIDS-free HIV-1-infected patients: a collaborative analysis of 18 HIV cohort studies. Lancet. 2009;373:1352–1363.
3. Kitahata MM, Gange SJ, Abraham AG, et al.. Effect of early versus deferred antiretroviral therapy for HIV on survival. N Engl J Med. 2009;360:1815–1826.
4. Sax PE, Baden LR. When to start antiretroviral therapy–ready when you are. N Engl J Med. 2009;360:1897–1899.
5. Vieillard V, Strominger JL, Debré P. NK cytotoxicity against CD4+ T cells during HIV-1 infection: a gp41 peptide induces the expression of an NKp44 ligand. Proc Natl Acad Sci U S A. 2005;102:10981–10986.
6. Vieillard V, Le Grand R, Dausset J, et al.. A new vaccine strategy against AIDS: HIV gp41 peptide immunization prevents NKp44L expression and CD4+ T cell depletion in SHIV-infected macaques. Proc Natl Acad Sci U S A. 2008;105:2100–2104.
7. Vieillard V, Costagliola D, Simon A, et al.. Specific adaptive humoral response against a gp41 motif inhibits CD4 T-cell sensitivity to NK lysis during HIV-1 infection. AIDS. 2006;20:1795–1804.
8. Madec Y, Boufassa F, Rouzioux C, et al.. Spontaneously controlled long-term viremia in HIV-1 seroconverters: an uncommon phenomenon? Clin Infect Dis. 2005;40:1350–1354.
9. Rouzioux C, Hubert JB, Burgard M, et al.. Early levels of HIV-1 DNA in peripheral blood mononuclear cells as a predictor of disease progression independent of HIV-1 RNA levels and CD4 cell counts. J Infect Dis. 2005;192:46–55.
10. Tilling R, Kinloch S, Goh LE, et al.. Parallel decline of CD8+/CD38++ T cells and viraemia in response to quadruple highly active antiretroviral therapy in primary HIV infection. AIDS. 2002;16:589–596.
11. Srinivasula S, Lempicki RA, Adelsberger JW, et al.. Differential effects of HIV viral load and CD4 count on proliferation of naive and memory CD4 and CD8 T lymphocytes. Blood. 2011;118:262–270.
12. Rodríguez B, Sethi AK, Cheruvu VK, et al.. Predictive value of plasma HIV RNA level on rate of CD4 T-cell decline in untreated HIV infection. JAMA. 2006;296:1498–1506.
13. Sodora DL, Allan JS, Apetrei C, et al.. Toward an AIDS vaccine: lessons from natural simian immunodeficiency virus infections of African nonhuman primate hosts. Nat Med. 2009;15:861–865.